Adaptors for nucleic acid constructs in transmembrane sequencing

ABSTRACT

The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/390,806, filed on Dec. 27, 2016, which is a continuation of U.S.application Ser. No. 13/147,159, filed on Nov. 15, 2011, which is anational stage filing under U.S.C. § 371 of PCT InternationalApplication No. PCT/GB2010/000160, which has an international filingdate of Jan. 29, 2010, and claims the benefit under 35 U.S.C. § 119(e)of U.S. Provisional Application Ser. No. 61/148,737, filed on Jan. 30,2009, the contents of each of which are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

The invention relates to adaptors for sequencing nucleic acids. Theadaptors may be used to generate single stranded constructs of nucleicacid for sequencing purposes. Such constructs may contain both strandsfrom a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) template. The invention also relates to the constructs generatedusing the adaptors, methods of making the adaptors and constructs, aswell as methods of sequencing double stranded nucleic acids.

BACKGROUND OF THE INVENTION

Stochastic detection is an approach to sensing that relies on theobservation of individual binding events between analyte molecules and areceptor. Stochastic sensors can be created by placing a single pore ofnanometer dimensions in an insulating membrane and measuringvoltage-driven ionic transport through the pore in the presence ofanalyte molecules. The frequency of occurrence of fluctuations in thecurrent reveals the concentration of an analyte that binds within thepore. The identity of an analyte is revealed through its distinctivecurrent signature, notably the duration and extent of current block(Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L.,Gouaux, J. E., and Bayley, H. (1997) Chem. Biol. 4, 497-505; and Bayley,H., and Cremer, P. S. (2001) Nature 413, 226-230).

Engineered versions of the bacterial pore forming toxin α-hemolysin(α-HL) have been used for stochastic sensing of many classes ofmolecules (Bayley, H., and Cremer, P. S. (2001) Nature 413, 226-230;Shin, S., H., Luchian, T., Cheley, S., Braha, O., and Bayley, H. (2002)Angew. Chem. Int. Ed. 41, 3707-3709; and Guan, X., Gu, L.-Q., Cheley,S., Braha, O., and Bayley, H. (2005) Chem Bio Chem 6, 1875-1881). In thecourse of these studies, it was found that attempts to engineer a-HL tobind small organic analytes directly can prove taxing, with rareexamples of success (Guan, X., Gu, L.-Q., Cheley, S., Braha, O., andBayley, H. (2005) Chem Bio Chem 6, 1875-1881). Fortunately, a differentstrategy was discovered, which utilised non-covalently attachedmolecular adaptors, notably cyclodextrins (Gu, L-Q., Braha, O., Conlan,S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690), but alsocyclic peptides (Sanchez-Quesada, J., Ghadiri, M. R., Bayley, H., andBraha, O. (2000) J Am. Chem. Soc. 122, 11758-11766) and cucurbiturils(Braha, O., Webb, J., Gu, L.-Q., Kim, K, and Bayley, H. (2005) Chem PhysChem 6, 889-892). Cyclodextrins become transiently lodged in the α-HLpore and produce a substantial but incomplete channel block. Organicanalytes, which bind within the hydrophobic interiors of cyclodextrins,augment this block allowing analyte detection (Gu, L.-Q., Braha, O.,Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690).

There is currently a need for rapid and cheap DNA or RNA sequencingtechnologies across a wide range of applications. Existing technologiesare slow and expensive mainly because they rely on amplificationtechniques to produce large volumes of nucleic acid and require a highquantity of specialist fluorescent chemicals for signal detection.Stochastic sensing has the potential to provide rapid and cheap DNAsequencing by reducing the quantity of nucleotide and reagents required.

SUMMARY OF THE INVENTION

The inventor(s) have surprisingly demonstrated that artificial,identifiable adaptors may be used to generate single stranded nucleicacid constructs that contain both strands of a double stranded nucleicacid template. The two strands of the template are covalently linked anddelineated (divided) by an adaptor. The adaptor not only allows thetransition point from one strand to the other strand to be identified,but also allows the construct to be purified before it is sequenced. Theadaptor may further allow the construct to be differentiated fromsimilar constructs in which the strands have a different source. Hence,the adaptors allow multiplex sequence analysis of templates originatingfrom separate individual sources.

The adaptors are particularly useful for sequencing double stranded DNA(dsDNA) and double stranded RNA (dsRNA). The adaptors may be used togenerate single stranded constructs containing both the sense andantisense strands of the dsDNA or dsRNA.

The adaptors are generally used in pairs. Both types of adaptor in thepair not only comprise a region of double stranded nucleic acid thatforms one half of a palindromic cleavage site, but also aredifferentially selectable from one another. Each pair comprises twotypes of adaptor; Type I and Type II. Type I adaptors comprise a hairpinloop, which allows covalent linkage of the two strands in a doublestranded nucleic acid template. Type II adaptors may comprise a hairpinloop, but do not have to. This combination of features allows thegeneration and purification of single stranded constructs in which bothstrands of a double stranded nucleic acid template are covalently linkedvia a Type I adaptor. Unwanted constructs formed by ligation of adaptorswith each other may be eliminated from the reaction mixture using thepalindromic cleavage site. Similarly, constructs containing one or otherof the two types of adaptor may be isolated from the reaction mixtureusing the adaptor's differential selectability.

Accordingly, the invention provides an adaptor for sequencing nucleicacids, which comprises a region of double stranded nucleic acid, whereinat least one end of the region forms one half of a palindromic cleavagesite and wherein the adaptor is differentially selectable from anotheradaptor. In some embodiments, the region is formed by hybridizationbetween two separated regions of a single stranded nucleic acid and theadaptor comprises a hairpin loop.

The invention also provides:

-   -   a pair of adaptors comprising an adaptor of the invention formed        by hybridization between two separated regions of a single        stranded nucleic acid and comprising a hairpin loop (Type I) and        an adaptor of the invention (Type II), wherein each type of        adaptor in the pair is differentially selectable from the other        type and wherein a complete palindromic cleavage site is formed        if any combination of the two types of adaptor are ligated to        one another;    -   a kit comprising at least two populations of adaptors of the        invention, wherein every adaptor in each population comprises a        nucleic acid sequence that is specific for the population;    -   a nucleic acid construct for use as a sequencing template        comprising a double stranded nucleic acid ligated to at least        one adaptor of the invention;    -   a single stranded nucleic acid construct for use as a sequencing        template comprising two strands of nucleic acid covalently        linked via an adaptor of the invention formed by hybridization        between two separated regions of a single stranded nucleic acid        and comprising a hairpin loop;    -   a circular nucleic acid construct for use as a sequencing        template comprising two strands of nucleic acid covalently        linked at each end via an adaptor of the invention formed by        hybridization between two separated regions of a single stranded        nucleic acid and comprising a hairpin loop;    -   a method for preparing an adaptor of the invention, comprising:    -   (a) providing two nucleic acids that are (i) capable of        hybridizing to one another to form one half of a palindromic        cleavage site and (ii) differentially selectable from those of        another adaptor; and    -   (b) contacting the nucleic acids under conditions which allow        them to hybridise and thereby preparing an adaptor;    -   a method for preparing an adaptor of the invention formed by        hybridization between two separated regions of a single stranded        nucleic acid and comprising a hairpin loop, comprising:    -   (a) providing a single stranded nucleic acid comprising (i) two        regions that are capable of hybridizing to one another, (ii) a        loop-forming region that is differentially selectable from that        of another adaptor and (iii) two ends which together form one        half of a palindromic cleavage site; and    -   (b) exposing the nucleic acid to conditions which allow the two        regions to hybridise and form a hairpin loop and thereby        preparing an adaptor;        -   a method for preparing a nucleic acid construct of the            invention, comprising:    -   (a) contacting at least one adaptor of the invention with two        strands of nucleic acid under conditions which allow ligation        between the adaptor(s) and the strands; and    -   (b) allowing the adaptor to ligate to the two strands and        thereby preparing a nucleic acid construct;        -   a method for preparing a single stranded nucleic acid            construct of the invention, comprising:    -   (a) contacting an adaptor of the invention formed by        hybridization between two separated regions of a single stranded        nucleic acid and comprising a hairpin loop with two strands of        nucleic acid under conditions which allow ligation between the        adaptor and the strands;    -   (b) allowing the adaptor to covalently link the two strands; and    -   (c) denaturing the covalently linked construct and thereby        preparing a single stranded nucleic acid construct;        -   a method for preparing a circular nucleic acid construct of            the invention, comprising:    -   (a) contacting at least two adaptors of the invention which        comprise a hairpin loop with two strands of nucleic acid under        conditions which allow ligation between the adaptors and        strands; and    -   (b) allowing an adaptor to covalently link the two strands at        each end and thereby preparing a circular nucleic acid        construct;        -   a method for preparing a sequence construct, comprising:    -   (a) providing double stranded nucleic acid;    -   (b) contacting the double stranded nucleic acid with a pair of        adaptors of the invention in which the Type I adaptors are not        capable of being cleaved or nicked and the Type II adaptors are        capable of being cleaved or nicked under conditions which allow        the adaptors to ligate to the nucleic acid;    -   (c) contacting the ligated products with a surface that        specifically binds the Type II adaptors and removing any unbound        products;    -   (d) contacting the surface with an enzyme that recognises the        complete palindromic cleavage site and removing any unbound        products;    -   (e) cleaving the Type II adaptors;    -   (f) contacting the soluble products produced in step (e) with a        surface that specifically binds the Type I adaptors and removing        any unbound products; and    -   (g) releasing from the surface the products remaining following        step (f) and thereby producing a sequencing construct;        -   a method of sequencing double stranded nucleic acid,            comprising:    -   (a) carrying out a method of the invention;    -   (b) denaturing the construct, if necessary, to form a single        stranded construct; and (c) sequencing the single stranded        construct and thereby sequencing the double stranded nucleic        acid; and        -   a kit for sequencing double stranded nucleic acid comprising            a pair of adaptors of the invention and means for cleaving            the palindromic cleavage sites.

DESCRIPTION OF THE FIGURES

FIG. 1 shows one embodiment of a Type I adaptor. The single stranded DNAstrand has self complementarity such that it will hybridise to itself,leaving a large hairpin loop of single stranded DNA, which is used toselectively bind the ‘Type I adaptor’ ligation products during thepurification. The terminus of the self hybridised adaptor encodes onehalf of the primary Restriction Endonuclease (arrowed, 1^(ry)), utilisedto cleave any ligation products created by adaptor:adaptor ligations,whether Type I:Type I, Type I:Type II or Type II:Type II.

FIG. 2 shows one embodiment of a Type II adaptor. In this Figure and allsubsequent Figures, the Type II adaptor comprises a hairpin loop. Thesingle stranded DNA is punctuated by a Biotin-dT base (starburst) whichwhen the strand self-hybridises, is presented in the single stranded‘bubble’ region. This biotin is a selectable characteristic of onlythose ligation products which include a Type II adaptor. The doublestranded element of this adaptor includes a recognition sequence of thesecondary Restriction Endonuclease, and (in common with the Type Iadaptor) is terminated with one half of the primary RestrictionEndonuclease recognition sequence, to enable elimination ofadaptor:adaptor ligation products, as previously.

FIG. 3 shows two types of hairpin adaptor (black; Type I and dark grey;Type II) are combined with blunt ended template dsDNA (light grey). BoxA shows the ideal situation where one Type I and one Type II adaptor areligated onto either end of an intervening template DNA sequence. Box Bdepicts that if there is no intervening template, an undesirableligation product is generated. The presence of a primary RE restrictionrecognition site (solid line box) within the ligated product is usefulfor the selective destruction of the undesirable ligation product. Analternative secondary RE restriction site (dotted box) within the TypeII adaptor is used to liberate the sequencing template (see below). ‘B’indicates the presence of a biotin moiety included upon the singlestranded element of the Type II adaptor.

FIG. 4 shows the generation of closed circular ‘DNA Dumbells’ commenceswith conventional random fragmentation of high molecular weight templateDNA. Only a proportion of the fragments generated will carry extendable3′OH underhang on both strands, which can be end repaired by DNApolymerase. A still smaller number of the repaired fragments willadditionally have 5′ PO₄ ends on both strands. Although small in number,any such blunt ended fragments will be receptive to the ligation ofartificial hairpin loop adaptors, which form the requisite closedcircular templates for exonuclease sequencing on both strands.

FIG. 5 shows the post-ligation of the Type I and Type II adaptors. Thedesired product for sequencing can be purified using the indicatedprocedure: Black lines represent ‘Type I’ adaptors; Dark Grey linesrepresent biotinylated ‘Type II’ adaptors; Light Grey lines indicatetemplate DNA. Crosshatched arrows indicate an operation without transferto a fresh plate. Empty arrows indicate transfer of the contents of theprevious well to a fresh plate. (1) Post ligation, the products arepipetted into an immobilised streptavidin plate. Only those ligationproducts harbouring a biotinylated Type II adaptor will bind. (2)Washing the plate will remove all Type I/Type I ligation products, etc.(3) Incubation with the ‘adaptor ligated to adaptor’ primary restrictionendonuclease will cleave the ‘adaptor/adaptor’ products. (4) Wash awayall of the restriction debris from the primary RE digestion. (5)Incubation with the ‘Type II adaptor’ encoded secondary restrictionendonuclease will cleave the bound Type II adaptor products. (6)Transfer the secondary RE digestion products to a fresh plate, ontowhich ssDNA complementary to the Type I single stranded hairpin ‘bubble’has been immobilised. Allow hybridisation of those RE fragments from 5to the immobilised ssDNA. (7) Wash away any unbound material, leavingthe only species retained as the desired ‘Type I adaptor ligated totemplate DNA’. (8) Using conditions which defeat the hybridisation ofthe ligation product to the immobilised DNA (heat, NaOH or any othermeans known in the art), transfer the desired product to a freshtube/plate for subsequent denaturation and sequencing.

FIG. 6 shows the treatment of the captured dumbbell structure (FIG. 1,A) with the enzyme encoded in the hybridised region of the Type IIadaptor releases a covalently closed structure as depicted here (left).Treatment of this structure with a denaturant yields a single strandedstructure (right) susceptible to exonuclease I digestion, which ifprocessive, will liberate nucleotides from the DNA to be interrogated,the linking artificial sequence nucleotides and then the reversecomplement nucleotides, which can be compared with the base callsalready made. Combination of the calls generates a consensus call ofgreater quality.

FIG. 7 shows an example of the single stranded product that is recoveredfrom the plate is digested by exonuclease to liberate 5′ monophosphatenucleosides that elicit a change in the current flow through an adaptormodified α-HL protein pore. The order in which the ‘bases’ are releasedand identified is sequential.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the polynucleotide sequence encoding one subunit ofwild type α-hemolysin (α-HL).

SEQ ID NO: 2 shows the amino acid sequence of one subunit of wild typeα-HL. Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216 and 219 to222 form α-helices. Amino acids 22 to 30, 35 to 44, 52 to 62, 67 to 71,76 to 91, 98 to 103, 112 to 123, 137 to 148, 154 to 159, 165 to 172, 229to 235, 243 to 261, 266 to 271, 285 to 286 and 291 to 293 formβ-strands. All the other non-terminal amino acids, namely 7 to 21, 31 to34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to242, 262 to 265, 272 to 274 and 287 to 290 form loop regions. Aminoacids 1 and 294 are terminal amino acids.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit ofα-HL M113R/N139Q (HL-RQ).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HLM113R/N139Q (HL-RQ). The same amino acids that form α-helices, β-strandsand loop regions in wild type α-HL form the corresponding regions inthis subunit.

SEQ ID NO: 5 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExoI) from E. coli.

SEQ ID NO: 6 shows the amino acid sequence of exonuclease I enzyme(EcoExoI) from E. coli. This enzyme performs processive digestion of 5′monophosphate nucleosides from single stranded DNA (ssDNA) in a 3′-5′direction. Amino acids 60 to 68, 70 to 78, 80 to 93, 107 to 119, 124 to128, 137 to 148, 165 to 172, 182 to 211, 213 to 221, 234 to 241, 268 to286, 313 to 324, 326 to 352, 362 to 370, 373 to 391, 401 to 454 and 457to 475 form α-helices. Amino acids 10 to 18, 28 to 26, 47 to 50, 97 to101, 133 to 136, 229 to 232, 243 to 251, 258 to 263, 298 to 302 and 308to 311 form β-strands. All the other non-terminal amino acids, 19 to 27,37 to 46, 51 to 59, 69, 79, 94 to 96102 to 106, 120 to 123, 129 to 132,149 to 164, 173 to 181, 212, 222 to 228 233, 242, 252 to 257, 264 to267, 287 to 297, 303 to 307, 312, 325, 353 to 361, 371, 372, 392 to 400,455 and 456, form loops Amino acids 1 to 9 are terminal amino acids. Theoverall fold of the enzyme is such that three regions combine to form amolecule with the appearance of the letter C, although residues 355-358,disordered in the crystal structure, effectively convert this C into anO-like shape. The amino terminus (1-206) forms the exonuclease domainand has homology to the DnaQ superfamily, the following residues(202-354) form an SH3-like domain and the carboxyl domain (359-475)extends the exonuclease domain to form the C-like shape of the molecule.Four acidic residues of EcoExoI are conserved with the active siteresidues of the DnaQ superfamily (corresponding to D15, E17, D108 andD186). It is suggested a single metal ion is bound by residues D15 and108. Hydrolysis of DNA is likely catalyzed by attack of the scissilephosphate with an activated water molecule, with H181 being thecatalytic residue and aligning the nucleotide substrate.

SEQ ID NO: 7 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 8 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides. Amino acids 19to 33, 44 to 61, 80 to 89, 103 to 111, 136 to 140, 148 to 163, 169 to183, 189 to 202, 207 to 217, 223 to 240, 242 to 252, 254 to 287, 302 to318, 338 to 350 and 365 to 382 form α-helices. Amino acids 36 to 40, 64to 68, 93 to 96, 116 to 120, 133 to 135, 294 to 297, 321 to 325, 328 to332, 352 to 355 and 359 to 363 form β-strands. All the othernon-terminal amino acids, 34, 35, 41 to 43, 62, 63, 69 to 79, 90 to 92,97 to 102, 112 to 115, 121 to 132, 141 to 147, 164 to 168, 184 to 188203to 206, 218 to 222, 241, 253, 288 to 293, 298 to 301, 319, 320, 326,327, 333 to 337, 351 to 358 and 364, form loops. Amino acids 1 to 18 and383 to 425 are terminal amino acids. The crystal structure has only beenresolved for the core domain of RecJ from Thermus thermophilus (residues40-463). To ensure initiation of translation and in vivo expression ofthe RecJ core domain a methionine residue was added at its aminoterminus, this is absent from the crystal structure information. Theresolved structure shows two domains, an amino (2-253) and a carboxyl(288-463) region, connected by a long α-helix (254-287). The catalyticresidues (D46, D98, H122, and D183) co-ordinate a single divalent metalion for nucleophilic attack on the phosphodiester bond. D46 and H120proposed to be the catalytic pair; however, mutation of any of theseconserved residues in the E. coli RecJ was shown to abolish activity.

SEQ ID NO: 9 shows the sequence of the I-SceI homing endonucleaserecognition site.

SEQ ID NO: 10 shows the nucleic sequence from which preferred nucleicacid linkers can be generated.

SEQ ID NO: 11 shows a preferred nucleic acid linker. MAL is maleimide.This linker is used in combination with SEQ ID NO: 14.

SEQ ID NO: 12 shows a preferred nucleic acid linker. MAL is maleimide.This linker is used in combination with SEQ ID NO: 15.

SEQ ID NO: 13 shows a preferred nucleic acid linker. MAL is maleimide.This linker is used in combination with SEQ ID NO: 16.

SEQ ID NO: 14 shows a preferred 15mer nucleic acid linker. MAL ismaleimide. This linker is complementary to and used in combination withSEQ ID NO: 11.

SEQ ID NO: 15 shows a preferred 15mer nucleic acid linker. MAL ismaleimide. This linker is complementary to and used in combination withSEQ ID NO: 12.

SEQ ID NO: 16 shows a preferred 15mer nucleic acid linker. MAL ismaleimide. This linker is complementary to and used in combination withSEQ ID NO: 13.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “aconstruct” includes “constructs”, reference to “a transmembrane pore”includes two or more such pores, reference to “a molecular adaptor”includes two or more such adaptors, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Adaptor

The invention provides adaptors for sequencing nucleic acids. Theadaptors comprise a region of double stranded nucleic acid. At least oneend of the region forms one half of a palindromic cleavage site. Theadaptors are differentially selectable from other adaptors. In someembodiments, the region is formed by hybridization between two separatedregions of a single stranded nucleic acid and the adaptors comprise ahairpin loop. Adaptors of the invention are typically used as part of apair of adaptors.

The adaptors of the invention have several advantages. The adaptorsfacilitate construction, purification and final release of a desiredsingle stranded sequencing construct, which comprises both strands of adouble stranded nucleic acid template. This ensures that, when theconstruct is sequenced, each position in the double stranded nucleicacid is not merely observed once, but is in fact interrogated twice.This gives greater certainty that each position in the nucleic acid hasbeen observed and that the aggregate call for both bases at eachposition is of a greater quality score than would be possible with asingle observation. In other words, the key advantage of the adaptors ofthe invention is that they allow each ‘base pair’ position of a doublestranded template to be effectively interrogated twice as part of thesame ‘read event’. This ensures that the quality of the sequencegenerated is consequently very much higher, with a reduced potential formisidentified base calls, or completely missed bases.

This is particularly helpful for the sequencing of dsDNA or dsRNA. Theadaptors of the invention allow the production of constructs containingboth the sense and antisense strands of dsDNA and dsRNA. Each ‘basepair’ position of the dsDNA or dsRNA can effectively be interrogatedtwice; once on the sense strand and once on the antisense strand.

This ability to interrogate each position twice is particularlyimportant when sequencing nucleic acids using stochastic sensing. Suchsequencing normally depends on the capture of every base in turn by thetransmembrane pore and a sufficiently high sampling rate to enableaccurate determination of the degree to which the current flowingthrough the pore is reduced. Being able to effectively interrogate everybase twice reduces the need to capture every base at a sufficiently highrate.

In addition, the adaptors of the invention allow the nucleic acid to beprovided in a form suitable for stochastic sensing. Only single strandednucleic acids can be threaded through transmembrane pores. In addition,many nucleic acid handling enzymes, which are an integral part of thesequencing methods described herein, are capable of only handling singlestranded nucleic acids.

The ability to interrogate each position twice is also helpful fordifferentiating between methylcytosine and thymine using stochasticsensing. These two bases result in very similar current traces when theypass through and interact with a transmembrane pore. It can therefore bedifficult to differentiate between the two. However, interrogation ofeach position in a nucleic acid twice will allow such differentiationbecause the complementary base for methylcytosine is guanine, whereasthe complementary base for thymine is adenine. Methylcytosine has ofcourse been linked with various diseases, including cancer.

Being artificial sequences, the adaptors of the invention have a greatdegree of flexibility in their actual sequence and thereforefunctionality can be built into the sequences used. For instance, anadaptor-specific sequence can be built into each adaptor. This allows aconstruct containing a particular adaptor to be differentiated from onecontaining a different adaptor. This is particularly helpful formultiplex sequence analysis of templates originating from separateindividual sources.

The adaptors are for sequencing nucleic acids. The adaptors arepreferably for sequencing a double stranded nucleic acid by generating asingle stranded nucleic acid construct that contains both strands of thedouble stranded nucleic acid template. The adaptors are more preferablyfor sequencing dsDNA or dsRNA by generating a single stranded nucleicacid construct that contains both the sense and antisense strands of thedsDNA or dsRNA.

Region of Double Stranded Nucleic Acid

The adaptors comprise a region of double stranded nucleic acid. Thepresence of this region means that the adaptors of the invention arecapable of ligating to other double stranded nucleic acids, such asdsDNA or dsRNA. The adaptors of the invention are also capable ofligating to themselves or other types of adaptors. As described in moredetail below, such ligation will result in the formation of a completepalindromic cleavable site. Suitable conditions that allow the ligationof the adaptors of the invention to double stranded nucleic acids orthemselves are discussed below.

The region of double stranded nucleic acid may comprise any type ofnucleic acid. A nucleic acid is a macromolecule comprising two or morenucleotides. The nucleic acid handled may comprise any combination ofany nucleotides. The nucleotides can be naturally occurring orartificial. A nucleotide typically contains a nucleobase, a sugar and atleast one phosphate group. The nucleobase is typically heterocyclic.Nucleobases include, but are not limited to, purines and pyrimidines andmore specifically adenine, guanine, thymine, uracil and cytosine. Thesugar is typically a pentose sugar. Nucleotide sugars include, but arenot limited to, ribose and deoxyribose. The nucleotide is typically aribonucleotide or deoxyribonucleotide. The nucleotide typically containsa monophosphate, diphosphate or triphosphate. Phosphates may be attachedon the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP,dAMP, dTMP, dGMP or dCMP.

The nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). The nucleic acid may include two strands of any synthetic nucleicacid known in the art, such as peptide nucleic acid (PNA), glycerolnucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid(LNA) or other synthetic polymers with nucleotide side chains. Whensequencing a double stranded nucleic acid template, the nucleic acid inthe adaptor is chosen such that the adaptors are capable of ligating tothe double stranded nucleic acid being sequenced.

The region of double stranded nucleic acid may be any length as long asthe palindromic cleavage site is functional when two adaptors ligatetogether. The region will typically be 40 or fewer base pairs, such as30 or fewer base pairs, 20 or fewer base pairs or 10 or fewer basepairs, in length. The region is preferably 5 to 20 base pairs in lengthand more preferably 6 to 10 base pairs in length.

The region may be formed by hybridization of two separate strands ofsingle stranded nucleic acid. The two separate strands may be the sametype of nucleic acid or different types of nucleic acid as long as theyhybridise. The two separate strands can be any of the types of nucleicacid described above. Suitable conditions that allow hybridization ofnucleic acids are discussed in more detail below.

The region of double stranded nucleic acid is preferably formed byhybridization of two separated regions of a single stranded nucleic acidsuch that the adaptor comprises a hairpin loop. In the context of theinvention, Type I adaptors comprise a hairpin loop. This allows Type Iadaptors to covalently link two strands of a double nucleic acidtemplate. Type II adaptors may or may not comprise a hairpin loop. It ispreferred that the Type II adaptors comprise a hairpin loop. Theformation of hairpin loops is known in the art. The hairpin loop istypically formed from single stranded nucleic acid. The hairpin loop maybe the same type of nucleic acid as that making up the region of doublestranded nucleic acid. Alternatively, the hairpin loop may be adifferent type of nucleic acid from that making up the region of doublestranded nucleic acid. The hairpin loop can be any of the types ofnucleic acid described above. As discussed in more detail below, thehairpin loop may be involved in the differential selectability of theadaptors of the invention. For instance, the hairpin loop may comprise aselectable binding moiety.

The hairpin loop may be any length. The hairpin loop is typically 50 orfewer bases, such as 40 or fewer bases, 30 or fewer bases, 20 or fewerbases or 10 or fewer bases, in length. The hairpin loop is preferablyfrom about 1 to 50, from 2 to 40 or from 6 to 30 bases in length. Longerlengths of the hairpin loop, such as from 15 to 50 bases, are preferredif the loop is involved in the differential selectability of theadaptor. Similarly, shorter lengths of the hairpin loop, such as from 1to 5 bases, are preferred if the loop is not involved in thedifferential selectability of the adaptor.

In adaptors without a hairpin loop, the region of double strandednucleic acid will have two free ends. One or both of these ends mayligate to a double stranded nucleic acid template. At least one endforms one half of a palindromic cleavage site. Both ends preferably formone half of the same palindromic cleavage site. One or both ends mayalso be involved in the differential selectability of the adaptors ofthe invention. Preferably, one end of the adaptor may ligate to a doublestranded nucleic acid template and forms one half of a palindromiccleavage site and the other end is involved in the differentialselectability of the adaptor.

In adaptors with a hairpin loop, the region of double stranded nucleicacid will have only one free end. The other end is closed by the hairpinloop. The free end not only forms one half of a palindromic cleavagesite, but also may ligate to a double stranded nucleic acid template.

The free end(s) of the region of double stranded nucleic acid may be inany form. The end(s) can be sticky. In other words, the end(s) do nothave to form a base pair. The sticky end(s) may have a 5′ or 3′overhang. It is preferred that the end(s) are blunt. In other words, itis preferred that the end(s) form a base pair. It is particularlypreferred that the end(s) of the region forming one half of apalindromic cleavage site are blunt.

In adaptors without a hairpin loop, it is preferred that the end thatligates to a double stranded nucleic acid template and forms one half ofa palindromic cleavage site is blunt and the other end that is involvedin the differential selectability of the adaptor is sticky.

One Half of a Palindromic Cleavage Site

A palindromic cleavage site is a palindromic consensus sequence in anucleic acid that may be cleaved in some manner. Several such sequencesare known in the art and may be used in the invention. Preferredpalindromic cleavage sites are shown below.

One half of a palindromic cleavage site is exactly one half of apalindromic consensus sequence. In other words, it is the amount of apalindromic cleavage site that when recombined with itself forms acomplete palindromic cleavage site. As discussed above, the ends formingthe one half of the palindromic cleavage site may be sticky or blunt.For instance, for a palindromic cleavage site having the followingsequence:

5′ . . . AAAATTTT . . . 3′ 3′ . . . TTTTAAAA . . . 5′one half of the palindromic cleavage site can be

5′ . . . AAAA . . . 3′ 3′ . . . TTTT . . . 5′ or 5′ . . . AAAAT . . . 3′3′ . . . TTT . . . 5′ or 5′ . . . AAA . . . 3′ 3′ . . . TTTTA . . . 5′

In the examples above, the first one half of the palindromic cleavagesite has blunt ends, while the second two one halves of the palindromiccleavage site have sticky ends.

As discussed above, the adaptors of the invention are typically used inpairs with one type of adaptor in the pair being differentiallyselectable from the other type of adaptor in the pair. Since both typesadaptors in the pair comprise one half a palindromic cleavage site, acomplete palindromic cleavage site is formed when one type of adaptorligates with an adaptor of the same type or of a different type. Forinstance, a complete palindromic cleavage site will be formed if Type Iligates to Type I (Type I:Type I), Type II ligates to Type II (TypeII:Type II) or Type I ligates to Type II (Type I:Type II). The formationof a complete palindromic cleavage site allows the ligated adaptors tobe cleaved. This is discussed in more detail below.

The complete palindromic cleavage site may be any length. For instance,palindromic cleavage sites are typically from 8 to 50 base pairs, suchas at least 10 base pairs, at least 12 base pairs, at least 14 basepairs, at least 16 base pairs, at least 20 base pairs, at least 30 basepairs or at least 40 base pairs, in length. For sequencing purposes, thelonger the palindromic cleavage site the better because the less likelythe sequence will appear randomly in an organism's genome. In acompletely random genome sequence (which of course is never found innature), a palindromic cleavage site of x base pairs in length would befound once every 4^(x) base pairs.

Preferred palindromic cleavage sites include restriction endonucleaserecognition sites. Restriction endonuclease recognition sites are sitesthat are cleaved by restriction endonuclease enzymes. Suitablerestriction endonuclease enzymes for use in the invention include, butare not limited to, those in Enzyme Classification (EC) groups 3.1.21.4and 3.1.21.5.

The restriction endonuclease recognition site may be a naturallyoccurring site that is cleaved by a naturally occurring restrictionendonuclease enzyme. Alternatively, the restriction endonucleaserecognition site and/or the restriction endonuclease may benon-naturally occurring. Engineering a restriction endonucleaserecognition site and/or a restriction endonuclease for use in theinvention offers various advantages. For instance, engineering anendonuclease to cleave a long and/or rare site means that theendonuclease is less likely to “accidentally” cleave one or more siteswith the double stranded nucleic acid template being interrogated.

Preferred restriction endonuclease recognition sites include, but arenot limited to, the following:

Sbfl 5′ . . . CCTGCAGG . . . 3′ 3′ . . . GGACGTCC . . . 5′ and AsiSI5′ . . . GCGATCGC . . . 3′ 3′ . . . CGCTAGCG . . . 5′

Preferred halves of these sites therefore include, but are not limitedto, the following:

Sbfl 5′ . . . CCTG . . . 3′ 3′ . . . GGAC . . . 5′ Sbfl5′ . . . CCT . . . 3′ 3′ . . . GGACG . . . 5′ Sbfl 5′ . . . AGG . . . 3′3′ . . . CGTCC . . . 5′ AsiSI 5′ . . . GCGA . . . 3′3′ . . . CGCT . . . 5′ AsiSI 5′ . . . CGC . . . 3′3′ . . . TAGCG . . . 5′ and AsiSI 5′ . . . GCG . . . 3′3′ . . . CGCTA . . . 5′Differential Selectability

Adaptors of the invention are differentially selectable from otheradaptors. Adaptors of the invention are differentially selectable fromdifferent types of adaptor of the invention. Type I adaptors aredifferentially selectable from Type II adaptors. Differentialselectability means that one type of adaptor can be delineated ordistinguished from another type of the adaptor on the basis of at leastone property. Any property may be used to differentially selectdifferent types of adaptors.

Generally, different types of adaptors are differentially selectablebecause they can be separated from each other. When used in pairs, eachtype of adaptor in the pair can be separated from the other type. Forinstance, Type I adaptors can be separated from Type II adaptors andvice versa. This facilitates the method of the invention discussed inmore detail below. Any means of separation can be used.

Differential selection preferably involves differential or selectivebinding to a surface. For instance, two types of adaptors of theinvention can of course be differentially selected if only one binds tosurface A and only the other binds to surface B. Adaptors of theinvention are therefore differentially selectable if they specificallybind to a surface. Adaptors specifically bind to a surface if they bindto the surface to a much greater degree than adaptors of a differenttype. In preferred embodiments, the adaptors bind to a surface to whichno other types of adaptor bind. Suitable surfaces are discussed in moredetail below.

It is most preferred that the adaptors can be separated from otheradaptors by differential binding. For instance, it is possible toseparate two types of adaptor (for example Types A and B) from eachother if the first type of adaptor (Type A) specifically binds to onesurface (surface A) and the second type of adaptor (Type B) binds toanother surface (surface B). A mixture of two types of adaptor willcontain unligated adaptors of both types, as well as ligated constructsof Type A:Type A, Type B:Type B and Type A:Type B. Contacting themixture with surface A will result in the binding of Type A adaptors andany constructs comprising a Type A adaptor. Similarly, contacting themixture with surface B will result in the binding of Type B adaptors andany constructs comprising a Type B adaptor. Ligated constructs can ofcourse be cleaved using the palindromic cleavage site.

The adaptors preferably comprise a selectable binding moiety. Aselectable binding moiety is a moiety that can be selected on the basisof its binding properties. Hence, a selectable binding moiety ispreferably a moiety that specifically binds to a surface. A selectablebinding moiety specifically binds to a surface if it binds to thesurface to a much greater degree than any other moiety used in theinvention. In preferred embodiments, the moiety binds to a surface towhich no other moiety used in the invention binds. If present, thehairpin loop preferably comprises the selective binding moiety.

Suitable selective binding moieties are known in the art. Preferredselective binding moieties include, but are not limited to, biotin, anucleic acid sequence, antibodies, antibody fragments, such as Fab andScSv, antigens, nucleic acid binding proteins, poly histidine tails andGST tags. The most preferred selective binding moieties are biotin and aselectable nucleic acid sequence. Biotin specifically binds to a surfacecoated with avidins. Selectable nucleic acid sequences specifically bind(i.e. hybridise) to a surface coated with homologous sequences. This isdiscussed in more detail below. Alternatively, selectable nucleic acidsequences specifically bind to a surface coated with nucleic acidbinding proteins. In the most preferred embodiment, one type of adaptorin a pair of adaptors comprises biotin and the other type of adaptorcomprises a selectable nucleic acid sequence.

Identification Sequences

In preferred embodiments, the adaptors comprise a nucleic acid sequencethat allows identification of the adaptor. The nucleic acid sequence maybe present in the region of double stranded nucleic acid or, if present,the hairpin loop.

The nucleic acid sequence is typically 12 or fewer bases, such as 10 orfewer bases, 8 or fewer bases or 6 or fewer bases, in length. Itcomprises a recognizable sequence that can be identified when aconstruct comprising the adaptor is sequenced in accordance with theinvention. In adaptors that comprising a hairpin loop, the sequence willbe identified as the adaptor part that links the two strands of nucleicacid to be interrogated is sequenced. In adaptors that lack a hairpinloop and are capable of being cleaved or nicked, the sequence istypically present between the end that ligates to the double strandednucleic acid template and the point at which adaptor can be cleaved ornicked. In such embodiments, the sequence remains ligated to the doublestranded nucleic acid template even once the adaptor is cleaved ornicked.

In preferred embodiments, the nucleic acid sequence identifies thesource of the two strands to which it is ligated. In such embodiments,the adaptor allows multiplex sequence analysis of templates originatingfrom separate individual sources. Each template is assigned a differentadaptor, each of which comprises a nucleic acid sequence that allowsidentification of the source of the template.

Adaptors that are Capable of being Cleaved or Nicked

In some embodiments, the adaptor is itself capable of being cleaved ornicked. In other words, the adaptor may be cleaved or nicked withouthaving to ligate to another adaptor. The region of double strandednucleic acid may be capable of being cleaved or nicked and/or, ifpresent, the hairpin loop may be capable of being cleaved or nicked. Inadaptors with a hairpin loop, it is preferred that the end of theadaptor that forms one half of a palindromic cleavage site (i.e. the endof the adaptor that ligates to the double stranded sequence template)can be separated from the selectable binding moiety. In adaptors withouta hairpin loop, it is preferred that one or both ends of the adaptor canbe separated from the selectable binding moiety.

Adaptors that are capable of being cleaved or nicked preferably containone or more, such as two, three or more, cleavage or nick sites. Anycleavage or nick site may be used in accordance with the invention. Suchsites include, but are not limited to, chemical cleavage or nick sites,RNA/DNA composite sites, non-natural bases (e.g. uracil) and restrictionendonuclease recognition sites and homing endonuclease recognitionsites.

Adaptors that are capable of being cleaved or nicked more preferablycomprise one or more restriction or homing endonuclease recognitionsites. It is preferred that the restriction or homing endonucleaserecognition site(s) are not the palindromic cleavage site formed if theadaptor ligates to another adaptor of the invention. Suitablerestriction or homing endonuclease recognition sites are known in theart. Preferred homing endonuclease recognition sites include, but arenot limited to, the following:

I-SceI  (SEQ ID NO: 9) 5′ . . . TAGGGATAACAGGGTAAT . . . 3′3′ . . . ATCCCTATTGTCCCATTA . . . 5′Pairs of Adaptors

The invention also provides pairs of adaptors of the invention. One typeof adaptor in the pair is formed by hybridization between two separatedregions of a single stranded nucleic acid and comprises a hairpin loop(Type I). The other type of adaptor in the pair may or may not have ahairpin loop (Type II). The Type II adaptor is preferably also formed byhybridization between two separated regions of a single stranded nucleicacid and comprises a hairpin loop. Each type of adaptor in the pair isdifferentially selectable from the other type. A complete palindromiccleavage site is formed if any combination of the two types of adaptorare ligated to one another. The adaptors may be any of those discussedabove.

It is preferred that the Type adaptor I can be separated from the TypeII adaptor and vice versa. Any method of separation described above canbe used. It is more preferred that the Type I adaptor can be separatedfrom the Type II adaptor by differential binding. It is even morepreferred that the Type I adaptor comprises a different selectablebinding moiety from the Type II adaptor. Preferably, the Type I adaptorcomprises a selectable nucleic acid and the Type II adaptor comprisesbiotin. All of these embodiments facilitate the method of the inventiondiscussed in more detail below.

It is also preferred that the Type I adaptor is not itself capable ofbeing cleaved or nicked and that the Type II is itself capable of beingcleaved or nicked. The Type II adaptor may be cleaved or nicked in anyof the ways discussed above.

It is further preferred that the Type I adaptor comprises a nucleic acidsequence that allows identification of the adaptor.

The most preferred pair of adaptors of the invention is summarised inTable 1 below.

Type I Type II Hairpin present Hairpin present Selectable nucleic acidBiotin Not itself capable of being cleaved or Itself capable of beingcleaved or nicked nicked Nucleic acid sequence that allows Nucleic acidsequence that allows identification of the adaptor identification of theadaptorKits

The invention also provides kits comprising at least two populations ofadaptors of the invention formed by hybridization between two separatedregions of a single stranded nucleic acid and comprising a hairpin loop(Type I). Every adaptor in each population comprises a nucleic acidsequence that is specific for the population. In other words, eachadaptor in a population comprises a sequence that allows the adaptor tobe identified as being part of that population and not part of one ofthe other populations. The two or more populations allow multiplexsequence analysis of double stranded nucleic acid templates originatingfrom two or more separate individual sources, such as from two or moreorganisms. Suitable organisms are discussed below. Each template isassigned a different population, each of which comprises a nucleic acidsequence that allows identification of the source of the template. Theidentifying nucleic acid sequence will be different in each population.The sequence is typically located at the same position in the adaptorsof each of the two or more populations. This allows efficientdifferentiation between the populations. Nucleic acid sequences thatallow identification of adaptors are discussed in more detail above. Anyof the embodiments discussed above are applicable to the kits of theinvention.

The kits may comprise any number of populations, such as 5, 10, 20, 50,100 or more populations.

The kits preferably further comprise two or more populations of Type IIadaptors such that every Type I adaptor forms a pair with a Type IIadaptor. Pairs of adaptors are discussed in more detail above. Any ofthe embodiments discussed above are applicable to the kits of theinvention.

The present invention also provides kits for sequencing double strandednucleic acid comprising a pair of adaptors of the invention and meansfor cleaving the palindromic cleavage site. The means is typically anenzyme as discussed above.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify, express and/or sequencepolynucleotide sequences, a membrane as defined above, a surface asdefined above or voltage or patch clamp apparatus. Reagents may bepresent in the kit in a dry state such that a fluid sample resuspendsthe reagents. The kit may also, optionally, comprise instructions toenable the kit to be used in the method of the invention or detailsregarding which patients the method may be used for. The kit may,optionally, comprise nucleotides.

Nucleic Acid Constructs

The present invention also provides nucleic acid constructs for use assequence templates. The constructs are useful for sequencing doublestranded nucleic acids. The constructs generally comprise two strands ofnucleic acid ligated to at least one adaptor of the invention. It istypically the sequence of the two strands of nucleic acid that needs tobe determined.

In one embodiment, the invention provides nucleic acid constructs foruse as a sequencing template comprising a double stranded nucleic acidligated to at least one adaptor of the invention. The construct maycomprise two adaptors, one ligated to each end of the double strandednucleic acid. The construct may comprise any of the adaptors discussedabove.

In another embodiment, the invention provides single stranded nucleicacid constructs for use as a sequencing template comprising two strandsof nucleic acid covalently linked via an adaptor of the invention formedby hybridization between two separated regions of a single strandednucleic acid and comprising a hairpin loop (Type I). The two strands aretypically derived from a double stranded nucleic acid, such as dsDNA ordsRNA. The construct may comprise any of the Type I adaptors discussedabove. Such constructs have several advantages as described above. Insome instances, it may be necessary to denature the construct to yield asingle stranded structure. Suitable conditions for denaturing nucleicacids are discussed in more detail below.

In a further embodiment, the invention provides circular nucleic acidconstructs comprising two strands of nucleic acid covalently linked ateach end via an adaptor of the invention formed by hybridization betweentwo separated regions of a single stranded nucleic acid and comprising ahairpin loop (Type I). The two strands are typically derived from adouble stranded nucleic acid, such as dsDNA or dsRNA. The construct maycomprise any of the Type I adaptors discussed above.

In all these embodiments, the two strands are preferably the sense andantisense strands of dsDNA or dsRNA.

Methods for Preparing Adaptors of the Invention

The invention also provides methods for preparing adaptors of theinvention. The methods involve providing two nucleic acids that are (i)capable of hybridizing to one another to form one half of a palindromiccleavage site and (ii) differentially selectable from those of anotheradaptor. These features are all discussed in detail above with referenceto the adaptors of the invention. The nucleic acids are contacted underconditions which allow them to hybridise and prepare an adaptor of theinvention. Such conditions are discussed in detail below.

The invention also provides methods for preparing Type I adaptors. Themethods involve providing a single stranded nucleic acid comprising (i)two regions that are capable of hybridizing to one another, (ii) aloop-forming region that is differentially selectable from that ofanother adaptor and (iii) two ends which together form one half of apalindromic cleavage site. These features are all discussed in detailabove with reference to the adaptors of the invention. The nucleic acidis exposed to conditions which allow the two regions to hybridise andform a hairpin loop and thereby prepare a Type I adaptor.

The nucleic acids or regions that are capable of hybridizing to oneanother preferably share at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% homology based on sequenceidentity. The nucleic acids or regions are more preferably complementary(i.e. share 100% homology based on sequence identity).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSP's containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two amino acidsequences would occur by chance. For example, a sequence is consideredsimilar to another sequence if the smallest sum probability incomparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Conditions that permit the hybridization are well-known in the art (forexample, Sambrook et al., 2001, Molecular Cloning: a laboratory manual,3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocolsin Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995)). Hybridization can be carriedout under low stringency conditions, for example in the presence of abuffered solution of 30 to 35% formamide, 1 M NaCl and 1% SDS (sodiumdodecyl sulfate) at 37° C. followed by a wash in from 1× (0.1650 M Na+)to 2× (0.33 M Na+) SSC (standard sodium citrate) at 50° C. Hybridizationcan be carried out under moderate stringency conditions, for example inthe presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and1% SDS at 37° C., followed by a wash in from 0.5× (0.0825 M Na+) to 1×(0.1650 M Na+) SSC at 55° C. Hybridization can be carried out under highstringency conditions, for example in the presence of a bufferedsolution of 50% formamide, 1 M NaCl, 1% SDS at 37° C., followed by awash in 0.1× (0.0165 M Na+) SSC at 60° C.

Methods for Preparing Constructs of the Invention

The invention also provides various methods for preparing the constructsof the invention. The constructs of the invention are discussed above.Any of the constructs of the invention can be made using these methods.

In one embodiment, the invention provides methods for preparing nucleicacid constructs of the invention. The methods involve contacting atleast one adaptor of the invention with two strands of nucleic acidunder conditions which allow ligation between the adaptor(s) and thestrands. Any of the adaptors discussed above may be used. The twostrands are typically derived from a double stranded nucleic acid, suchas dsDNA or dsRNA. Conditions suitable for ligating nucleic acids areknown in the art. Such conditions include, but are not limited to, 50 mMTris-HCl, 10 mM MgCl₂, 1 mM ATP, 10 mM Dithiothreitol, pH 7.5 and 25° C.The adaptor(s) are then allowed to ligate to the two strands and therebyprepare a nucleic acid construct.

In another embodiment, the invention provides methods for preparingsingle stranded nucleic acid constructs of the invention. The methodsinvolve contacting a Type I adaptor with two strands of nucleic acidunder conditions which allow ligation between the adaptor and thestrands. Any of the Type I adaptors discussed above may be used. The twostrands are typically derived from a double stranded nucleic acid, suchas dsDNA or dsRNA. Conditions suitable for ligating nucleic acids arediscussed above. The adaptor is allowed to covalently link the twostrands at each end. The covalently linked constructs are then denaturedto prepare single stranded nucleic acid constructs. Suitable conditionsfor denaturing nucleic acids include, but are not limited to, pH,temperature and ionic strength.

In yet another embodiment, the invention provides method for preparingcircular nucleic acid constructs of the invention. The methods involvecontacting at least two Type I adaptors with two strands of nucleic acidunder conditions which allow ligation between the adaptors and strands.The at least two Type I adaptors may be the same or different. Any ofthe Type I adaptors discussed above may be used. The two strands aretypically derived from a double stranded nucleic acid, such as dsDNA ordsRNA. Conditions suitable for ligating nucleic acids are discussedabove. An adaptor is then allowed to covalently link the two strands ateach end and thereby prepare circular nucleic acid constructs.

In yet another embodiment, the invention provides methods for preparingsequence constructs. The methods prepare single stranded nucleic acidconstructs comprising the two strands of a double stranded nucleic acidcovalently linked via a Type I adaptor. The methods involve providingdouble stranded nucleic acid. The providing preferably involves randomlyfragmenting template nucleic acid. The ends of the double strandednucleic acid may be repaired to form blunt ends. Any of the nucleicacids disclosed above can be used. The methods are typically carried outusing a double stranded nucleic acid whose sequence is unknown.Alternatively, the methods may be carried out using a double strandednucleic acid whose sequence is known or can be predicted.

The methods may be carried out in vitro on double stranded nucleic acidobtained from or extracted from any organism or microorganism. Theorganism or microorganism is typically prokaryotic, eukaryotic or anarcheeon and typically belongs to one the five kingdoms: plantae,animalia, fungi, monera and protista. The methods may be carried out invitro on double stranded nucleic acid obtained from or extracted fromany virus. Typically, the double stranded nucleic acid is human inorigin, but alternatively it may be from another mammal animal such asfrom commercially farmed animals such as horses, cattle, sheep or pigsor may alternatively be pets such as cats or dogs.

The double stranded nucleic acid is typically processed prior toundergoing the methods, for example by centrifugation or by passagethrough a membrane that filters out unwanted molecules or cells, such asred blood cells. The double stranded nucleic acid may be usedimmediately upon being taken. The double stranded nucleic acid may alsobe typically stored prior to undergoing the methods, preferably below−70° C.

The double stranded nucleic acid is preferably dsDNA or dsRNA.

The double stranded nucleic acid is contacted with a pair of Type I andType II adaptors of the invention under conditions which allow theadaptors to ligate to the nucleic acid. The Type II adaptor is itselfcapable of being cleaved or nicked as discussed above. Conditionssuitable for ligating nucleic acids are discussed above. Suitable pairsof Type I adaptors and Type II adaptors are also discussed above.

The ligated products are then contacted with a surface that specificallybinds the Type II adaptors that are capable of being cleaved or nicked.Any constructs containing Type II adaptors will bind to the surface.Suitable surfaces include, but are not limited to, metal (gold inparticular), agarose, dextran, polystyrene, glass, silica (bonded andunbonded) and cellulose. Preferably, the surface specifically binds aselectable binding moiety on the Type II adaptors. The surface is mostpreferably coated with avidins.

Any unbound products are then removed. This is typically done by washingthe surface with a suitable buffer. Suitable buffers include, but arenot limited to, Tris, HEPES and MOPS at suitable ionic concentrations.This step removes all constructs formed by the ligation of a Type Iadaptor to a Type I adaptor (Type I:Type I).

The surface is then contacted with an enzyme that recognises thecomplete palindromic cleavage site. Suitable enzymes are discussedabove. This step will cleave any remaining (i e bound) constructs formedfrom the ligation of an adaptor to an adaptor, i.e. Type I:Type II orType II:Type II.

Again, any unbound products are removed, typically by washing. This stepensures that only Type II adaptors in isolation or constructs containingthe double stranded nucleic acid and at least one Type II adaptor remainbound to the surface.

The Type H adaptors are then cleaved. Methods for doing this arediscussed above. This step ensures the release of the constructscontaining the double stranded nucleic acid and at least one Type IIadaptor from the surface.

The soluble products are then contacted with a surface that specificallybinds the Type I adaptors that are not capable of being cleaved ornicked. Any remaining constructs containing Type I adaptors bind to thesurface. Each construct will contain the double stranded nucleic acidcovalently linked at one end via a Type I adaptor. The surfacepreferably specifically binds a selectable binding moiety on the Type Iadaptors. The surface is more preferably coated with nucleic acidsequences that are at least 80%, such as least 90%, at least 95% or atleast 99%, homologous based on sequence identity to a selectable nucleicacid sequence in the Type I adaptors. The surface is most preferablycoated with nucleic acid sequences that are complementary to aselectable nucleic acid sequence in the Type I adaptors. Again, unboundproducts are removed.

Finally, any remaining products are released from the surface. Thosereleased products represent a sequencing construct of the invention inwhich a double stranded nucleic acid is covalently linked at one end viaa Type I adaptor. The construct may also contain fragments of the TypeII adaptor at the ends of the double stranded nucleic acid.

The resulting construct may need to be denatured to form a singlestranded construct. Conditions suitable for denaturing double strandednucleic acids are discussed above.

Methods of Sequencing Double Stranded Nucleic Acid

The invention also provides methods of sequencing double strandednucleic acid. The methods involve carrying out one of the methodsdescribed above for preparing nucleic acid constructs. The constructcontains two strands of nucleic acid, preferably DNA or RNA, covalentlylinked via a Type I adaptor. If necessary, the construct is denatured toform a single stranded construct. Conditions for doing this aredescribed above.

The single stranded construct is then be sequenced. Sequencing thesingle stranded construct will provide the sequence of, in order, onestrand, the Type I adaptor and the other strand. The strands will ofcourse be in opposite orientations. In some embodiments, fragments ofthe Type II adaptors may also be present in the single stranded nucleicacid construct.

The methods of the invention are advantageous because each position inthe double stranded nucleic acid is interrogated twice (i.e. once oneach strand). The methods preferably involve sequencing double strandednucleic acid containing or suspected of containing methylcytosine. Ifthe Type I adaptor comprises a nucleic acid sequence that identifies thesource of the double stranded nucleic acid, this will also be recognisedusing the methods of the invention.

The whole or only part of the construct may be sequenced using thesemethods. The construct can be any length. For example, the construct canbe at least 10, at least 50, at least 100, at least 150, at least 200,at least 250, at least 300, at least 400 or at least 500 nucleotides inlength. The methods are typically carried out in vitro.

By effectively doubling the interrogation of every base, the inventionmay improve the data quality of all existing second generationsequencing chemistries and next generation sequencing technologies indevelopment. Any method of sequencing the single stranded nucleic acidconstruct may be used in accordance with the invention. Suitable methodsare known in the art. Such methods include, but are not limited to,Sanger (or dideoxy) method, the Maxam-Gilbert (chemical cleavage)method, Life Technologies' SOLiD (which uses sequencing by ligation),Illumina Genome Analyser (which uses fluorescent reversible terminatorchemistry on amplified templates), 454 Genome Sequencer FLX (which usespyrosequencing chemistry on amplified templates), Helicos Heliscope(which uses true single molecule sequencing by fluorescent reversibleterminator chemistry on unamplified (adapter modified) templates),Bionanomatrix (electronic discrimination of bases in etched channels),Danaher Motion (‘polony’ sequencing), LingVitae (‘design polymer’sequencing), Pacific BioSciences' Single Molecule Sequencing byfluorescent nucleotide DNA polymerization and Visigen's (Sequencing byFRET interaction of donor and acceptor during a DNA polymerisationreaction).

There are also a number of ways that transmembrane pores can be used tosequence nucleic acid molecules. One way involves the use of anexonuclease enzyme, such as a deoxyribonuclease. In this approach, theexonuclease enzyme is used to sequentially detach the nucleotides from atarget nucleic strand. The nucleotides are then detected anddiscriminated by the pore in order of their release, thus reading thesequence of the original strand.

Another way of sequencing nucleic acids involves the use of an enzymethat pushes or pulls the target nucleic acid strand through the pore incombination with an applied potential. In this approach, the ioniccurrent fluctuates as a nucleotide in the target strand passes throughthe pore. The fluctuations in the current are indicative of the sequenceof the strand.

A third way of sequencing a nucleic acid strand is to detect thebyproducts of a polymerase in close proximity to a pore detector. Inthis approach, nucleoside phosphates (nucleotides) are labelled so thata phosphate labelled species is released upon the addition of apolymerase to the nucleotide strand and the phosphate labelled speciesis detected by the pore. The phosphate species contains a specific labelfor each nucleotide. As nucleotides are sequentially added to thenucleic acid strand, the bi-products of the base addition are detected.The order that the phosphate labelled species are detected can be usedto determine the sequence of the nucleic acid strand.

Any of these three methods can be used to sequence in accordance withthe invention.

In one preferred embodiment, the sequencing is carried out by methodscomprising (i) contacting the construct with a transmembrane pore havingan exonuclease and a molecular adaptor covalently attached thereto sothat the exonuclease digests an individual nucleotide from one end ofthe construct; (ii) contacting the nucleotide with the pore so that thenucleotide interacts with the molecular adaptor; (iii) measuring thecurrent passing through the pore during the interaction and therebydetermining the identity of the nucleotide; and (iv) repeating steps (i)to (iii) at the same end of the construct and thereby determining thesequence of the target sequence. Hence, the methods involve stochasticsensing of a proportion of the nucleotides in the construct in asuccessive manner in order to sequence the construct. Individualnucleotides are described below.

In another preferred embodiment, the sequencing is carried out bymethods comprising (i) contacting the construct with a transmembranepore having a nucleic acid handling enzyme attached thereto so that theenzyme pushes or pulls the construct through the pore and a proportionof the nucleotides in the construct interacts with the pore and (ii)measuring the current passing through the pore during each interactionand thereby determining the sequence of the construct. Hence, themethods involve stochastic sensing of a proportion of the nucleotides ina construct as the nucleotides pass through the barrel or channel in asuccessive manner in order to sequence the construct.

Transmembrane Pores

A transmembrane pore is a pore that permits ions driven by an appliedpotential to flow from one side of a membrane to the other side of themembrane. The pore preferably permits nucleotides to flow from one sideof a membrane to the other along the applied potential. The porepreferably allows a nucleic acid, such as DNA or RNA, to be pushed orpulled through the pore.

The pore is preferably a transmembrane protein pore. A transmembraneprotein pore is a polypeptide or a collection of polypeptides thatpermits ions driven by an applied potential to flow from one side of amembrane to the other side of the membrane.

The pore may be isolated, substantially isolated, purified orsubstantially purified. A pore is isolated or purified if it iscompletely free of any other components, such as lipids or other pores.A pore is substantially isolated if it is mixed with carriers ordiluents which will not interfere with its intended use. For instance, apore is substantially isolated or substantially purified if it presentin a form that comprises less than 10%, less than 5%, less than 2% orless than 1% of other components, such as lipids or other pores. Thepore is typically present in a lipid bilayer.

The pore may be a monomer or an oligomer. The pore is preferably made upof several repeating subunits, such as 6, 7 or 8 subunits. The pore ismore preferably a heptameric pore. The pore typically comprises a barrelor channel through which the ions may flow. The subunits of the poretypically surround a central axis and contribute strands to atransmembrane β barrel or channel or a transmembrane α-helix bundle orchannel.

The barrel or channel of the pore typically comprises amino acids thatfacilitate interaction with nucleotides or nucleic acids. These aminoacids are preferably located near a constriction of the barrel orchannel. The pore typically comprises one or more positively chargedamino acids, such as arginine, lysine or histidine. These amino acidstypically facilitate the interaction between the pore and nucleotides ornucleic acids. The nucleotide detection can be facilitated with anadaptor. This is discussed in more detail below.

Pores for use in accordance with the invention can be β-barrel pores,α-helix bundle pores or solid state pores. β-barrel pores comprise abarrel or channel that is formed from β-strands. Suitable β-barrel poresinclude, but are not limited to, β-toxins, such as α-hemolysin, anthraxtoxin and leukocidins, and outer membrane proteins/porins of bacteria,such as Mycobacterium smegmatis porin A (MspA), outer membrane porin F(OmpF), outer membrane porin G (OmpG), outer membrane phospholipase Aand Neisseria autotransporter lipoprotein (NalP). α-helix bundle porescomprise a barrel or channel that is formed from α-helices. Suitableα-helix bundle pores include, but are not limited to, inner membraneproteins and α outer membrane proteins, such as WZA.

Suitable solid state pores include, but are not limited to, siliconnitride pores, silicon dioxide pores and graphene pores. Other suitablesolid state pores and methods of producing them are discussed in U.S.Pat. No. 6,464,842, WO 03/003446, WO 2005/061373, U.S. Pat. Nos.7,258,838, 7,466,069, 7,468,271 and 7,253,434.

The pore is preferably derived from α-hemolysin (α-HL). The wild typeα-HL pore is formed of seven identical monomers or subunits (i.e. it isheptameric). The sequence of one wild type monomer or subunit ofα-hemolysin is shown in SEQ ID NO: 2. The pore preferably comprisesseven subunits of the sequence shown in SEQ ID NO: 2 or a variantthereof. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to290 and 294 of SEQ ID NO: 2 form loop regions. Residues 113 and 147 ofSEQ ID NO: 2 form part of a constriction of the barrel or channel ofα-HL.

A variant of SEQ ID NO: 2 is a subunit that has an amino acid sequencewhich varies from that of SEQ ID NO: 2 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into a membrane along with other appropriate subunits and itsability to oligomerise to form a pore may be determined.

The variant may include modifications that facilitate covalentattachment to or interaction with the nucleic acid handling enzyme. Thevariant preferably comprises one or more reactive cysteine residues thatfacilitate attachment to the enzyme. For instance, the variant mayinclude a cysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45,50, 51, 237, 239 and 287 and/or on the amino or carboxy terminus of SEQID NO: 2. Preferred variants comprise a substitution of the residue atposition 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 2 with cysteine (K8C,T9C, N17C, K237C, S239C or E287C).

The variant may be modified to facilitate genetic fusion of the enzyme.For instance, one or more residues adjacent to the insertion site may bemodified, such as deleted, to facilitate insertion of the enzyme and/orlinkers. If the enzyme is inserted into loop 2 of SEQ ID NO: 2, one ormore of residues D45, K46, N47, H48, N49 and K50 of SEQ ID NO: 2 may bedeleted.

The variant may also include modifications that facilitate anyinteraction with nucleotides or facilitate orientation of a molecularadaptor as discussed below. The variant may also contain modificationsthat facilitate covalent attachment of a molecular adaptor.

In particular, the variant preferably has a glutamine at position 139 ofSEQ ID NO: 2. The variant preferably has an arginine at position 113 ofSEQ ID NO: 2. The variant preferably has a cysteine at position 119, 121or 135 of SEQ ID NO: 2. SEQ ID NO: 4 shows the sequence of SEQ ID NO: 2except that it has an arginine at position 113 (M113R) and a glutamineat position 139 (N139Q). SEQ ID NO: 4 or a variant thereof may be usedto form a pore in accordance with the invention.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium, orexpressed recombinantly by a bacterium such as Escherichia coli.Variants also include non-naturally occurring variants produced byrecombinant technology. Over the entire length of the amino acidsequence of SEQ ID NO: 2 or 4, a variant will preferably be at least 50%homologous to that sequence based on amino acid identity. Morepreferably, the variant polypeptide may be at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% and more preferably at least 95%, 97% or 99% homologous basedon amino acid identity to the amino acid sequence of SEQ ID NO: 2 or 4over the entire sequence. There may be at least 80%, for example atleast 85%, 90% or 95%, amino acid identity over a stretch of 200 ormore, for example 230, 250, 270 or 280 or more, contiguous amino acids(“hard homology”).

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 or 4 in addition to those discussed above, for example up to 1,2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions maybe made, for example, according to Table 2 below.

TABLE 2 Conservative substitutions Amino acids in the same block in thesecond column and preferably in the same line in the third column may besubstituted for each other. NON-AROMATIC Non-polar G A P I L V Polar -uncharged C S T M N Q Polar - charged D E H K R AROMATIC H F W Y

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may fragments of SEQ ID NO: 2 or 4. Such fragments retain poreforming activity. Fragments may be at least 50, 100, 200 or 250 aminoacids in length. A fragment preferably comprises the pore forming domainof SEQ ID NO: 2 or 4. Fragments typically include residues 119, 121,135, 113 and 139 of SEQ ID NO: 2 or 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 2 or 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 2 or 4 is a subunit that hasan amino acid sequence which varies from that of SEQ ID NO: 2 or 4 andwhich retains its ability to form a pore. A variant typically containsthe regions of SEQ ID NO: 2 or 4 that are responsible for poreformation. The pore forming ability of α-HL, which contains a β-barrel,is provided by β-strands in each subunit. A variant of SEQ ID NO: 2 or 4typically comprises the regions in SEQ ID NO: 2 that form β-strands. Theamino acids of SEQ ID NO: 2 or 4 that form β-strands are discussedabove. One or more modifications can be made to the regions of SEQ IDNO: 2 or 4 that form β-strands as long as the resulting variant retainsits ability to form a pore. Specific modifications that can be made tothe β-strand regions of SEQ ID NO: 2 or 4 are discussed above.

A variant of SEQ ID NO: 2 or 4 preferably includes one or moremodifications, such as substitutions, additions or deletions, within itsα-helices and/or loop regions. Amino acids that form α-helices and loopsare discussed above.

The variant may be modified for example by the addition of histidine oraspartic acid residues to assist its identification or purification orby the addition of a signal sequence to promote their secretion from acell where the polypeptide does not naturally contain such a sequence.

The pore may be labelled with a revealing label. The revealing label maybe any suitable label which allows the pore to be detected. Suitablelabels include, but are not limited to, fluorescent molecules,radioisotopes, e.g. ¹²⁵I, ³⁵S, ¹⁴C, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

The pore may be isolated from a pore producing organism, such asStaphylococcus aureus, or made synthetically or by recombinant means.For example, the pore may be synthesised by in vitro translation andtranscription. The amino acid sequence of the pore may be modified toinclude non-naturally occurring amino acids or to increase the stabilityof the pore. When the pore is produced by synthetic means, such aminoacids may be introduced during production. The pore may also be alteredfollowing either synthetic or recombinant production.

The pore may also be produced using D-amino acids. For instance, thepores may comprise a mixture of L-amino acids and D-amino acids. This isconventional in the art for producing such proteins or peptides.

The pore may also contain other non-specific chemical modifications aslong as they do not interfere with its ability to form a pore. A numberof non-specific side chain modifications are known in the art and may bemade to the side chains of the pores. Such modifications include, forexample, reductive alkylation of amino acids by reaction with analdehyde followed by reduction with NaBH₄, amidination withmethylacetimidate or acylation with acetic anhydride. The modificationsto the pore can be made after expression of each subunit or after thesubunits have been used to form a pore.

The pore can be produced using standard methods known in the art.Polynucleotide sequences encoding a pore or pore subunit may be isolatedand replicated using standard methods in the art. Polynucleotidesequences encoding a pore or pore subunit may be expressed in abacterial host cell using standard techniques in the art. The pore maybe produced in a cell by in situ expression of the polypeptide from arecombinant expression vector. The expression vector optionally carriesan inducible promoter to control the expression of the polypeptide.

A pore may be produced in large scale following purification by anyprotein liquid chromatography system from pore producing organisms orafter recombinant expression as described below. Typical protein liquidchromatography systems include FPLC, AKTA systems, the Bio-Cad system,the Bio-Rad BioLogic system and the Gilson HPLC system.

Nucleic Acid Handling Enzyme

A nucleic acid handling enzyme is a polypeptide that is capable ofinteracting with and modifiying at least one property of a nucleic acid.The enzyme may modify the nucleic acid by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The enzyme may modify the nucleic acid by orienting itor moving it to a specific position. Any of the nucleic acids discussedabove may be handled by the enzyme.

The nucleic acid handled by the enzyme is preferably single stranded.The nucleic acid handled by the enzyme may be double stranded, such asdsDNA or dsRNA. Enzymes that handle single stranded nucleic acids may beused to sequence double stranded DNA as long as the double stranded DNAis chemically or thermally dissociated into a single strand before it ishandled by the enzyme.

It is preferred that the tertiary structure of the nucleic acid handlingenzyme is known. Knowledge of the three dimensional structure of theenzyme allows modifications to be made to the enzyme to facilitate itsfunction in the methods of the invention.

The enzyme may be any size and have any structure. For instance, theenzyme may be an oligomer, such as a dimer or trimer. The enzyme ispreferably a small, globular polypeptide formed from one monomer. Suchenzymes are easy to handle and are less likely to interfere with thepore forming ability of the pore or pore subunit, particularly if fusedto or inserted into the sequence of the pore or pore subunit.

The amino and carboxy terminii of the enzyme are preferably in closeproximity. The amino and carboxy terminii of the enzyme are morepreferably presented on same face of the enzyme. Such embodimentsfacilitate insertion of the enzyme into the sequence of the pore or poresubunit. For instance, if the amino and carboxy terminii of the enzymeare in close proximity, each can be attached by genetic fusion toadjacent amino acids in the sequence of the pore or pore subunit.

It is also preferred that the location and function of the active siteof the enzyme is known. This prevents modifications being made to theactive site that abolish the activity of the enzyme. It also allows theenzyme to be attached to the pore so that the enzyme handles theconstruct in such a way that a proportion of the nucleotides in theconstruct interacts with the pore. It is beneficial to position theactive site of the enzyme as close as possible to the part of the porethat forms part of the opening of the barrel of channel of the pore,without the enzyme itself presenting a block to the flow of current.Knowledge of the way in which an enzyme may orient nucleic acids alsoallows an effective pore-enzyme construct to be designed.

In order that most of the nucleotides in the construct are correctlyidentified by stochastic sensing, the enzyme must handle the nucleicacid in a buffer background which is compatible with discrimination ofthe nucleotides. The enzyme preferably has at least residual activity ina salt concentration well above the normal physiological level, such asfrom 100 mM to 2000 mM. The enzyme is more preferably modified toincrease its activity at high salt concentrations. The enzyme may alsobe modified to improve its processivity, stability and shelf life.

Suitable modifications can be determined from the characterisation ofnucleic acid handling enzymes from extremphiles such as halophilic,moderately halophilic bacteria, thermophilic and moderately thermophilicorganisms, as well as directed evolution approaches to altering the salttolerance, stability and temperature dependence of mesophilic orthermophilic exonucleases.

The enzyme also preferably retains at least partial activity attemperatures from 10° C. to 60° C., such as at room temperature. Thisallows the construct to sequence nucleic acids at a variety oftemperatures, including room temperature.

The nucleic acid handling enzyme is preferably a nucleolytic enzyme. Thenucleic acid handling enzyme is more preferably member of any of theEnzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Thenucleic acid handling enzyme is more preferably any one of the followingenzymes:

-   -   3.1.11.-Exodeoxyribonucleases producing 5′-phosphomonoesters.        -   3.1.11.1 Exodeoxyribonuclease I.        -   3.1.11.2 Exodeoxyribonuclease III.        -   3.1.11.3 Exodeoxyribonuclease (lambda-induced).        -   3.1.11.4 Exodeoxyribonuclease (phage SP3-induced).        -   3.1.11.5 Exodeoxyribonuclease V.        -   3.1.11.6 Exodeoxyribonuclease VII.    -   3.1.13.-Exoribonucleases producing 5′-phosphomonoesters.        -   3.1.13.1 Exoribonuclease II.        -   3.1.13.2 Exoribonuclease H.        -   3.1.13.3 Oligonucleotidase.        -   3.1.13.4 Poly(A)-specific ribonuclease.        -   3.1.13.5 Ribonuclease D.    -   3.1.14.-Exoribonucleases producing 3′-phosphomonoesters.        -   3.1.14.1 Yeast ribonuclease.    -   3.1.15.-Exonucleases active with either ribo- or        deoxyribonucleic acid producing 5′ phosphomonoesters        -   3.1.15.1 Venom exonuclease.    -   3.1.16.-Exonucleases active with either ribo- or        deoxyribonucleic acid producing 3′ phosphomonoesters        -   3.1.16.1 Spleen exonuclease.    -   3.1.21.-Endodeoxyribonucleases producing 5′-phosphomonoesters.        -   3.1.21.1 Deoxyribonuclease I.        -   3.1.21.2 Deoxyribonuclease IV (phage-T(4)-induced).        -   3.1.21.3 Type I site-specific deoxyribonuclease.        -   3.1.21.4 Type II site-specific deoxyribonuclease.        -   3.1.21.5 Type III site-specific deoxyribonuclease.        -   3.1.21.6 CC-preferring endodeoxyribonuclease.        -   3.1.21.7 Deoxyribonuclease V.    -   3.1.22.-Endodeoxyribonucleases producing other than        5′-phosphomonoesters.        -   3.1.22.1 Deoxyribonuclease II.        -   3.1.22.2 Aspergillus deoxyribonuclease K(1).        -   3.1.22.3 Transferred entry: 3.1.21.7.        -   3.1.22.4 Crossover junction endodeoxyribonuclease.        -   3.1.22.5 Deoxyribonuclease X.    -   3.1.25.-Site-specific endodeoxyribonucleases specific for        altered bases.        -   3.1.25.1 Deoxyribonuclease (pyrimidine dimer).        -   3.1.25.2 Transferred entry: 4.2.99.18.    -   3.1.26.-Endoribonucleases producing 5′-phosphomonoesters.        -   3.1.26.1 Physarum polycephalum ribonuclease.        -   3.1.26.2 Ribonuclease alpha.        -   3.1.26.3 Ribonuclease III.        -   3.1.26.4 Ribonuclease H.        -   3.1.26.5 Ribonuclease P.        -   3.1.26.6 Ribonuclease IV.        -   3.1.26.7 Ribonuclease P4.        -   3.1.26.8 Ribonuclease M5.        -   3.1.26.9 Ribonuclease (poly-(U)-specific).        -   3.1.26.10 Ribonuclease IX.        -   3.1.26.11 Ribonuclease Z.    -   3.1.27.-Endoribonucleases producing other than        5′-phosphomonoesters.        -   3.1.27.1 Ribonuclease T(2).        -   3.1.27.2 Bacillus subtilis ribonuclease.        -   3.1.27.3 Ribonuclease T(1).        -   3.1.27.4 Ribonuclease U(2).        -   3.1.27.5 Pancreatic ribonuclease.        -   3.1.27.6 Enterobacter ribonuclease.        -   3.1.27.7 Ribonuclease F.        -   3.1.27.8 Ribonuclease V.        -   3.1.27.9 tRNA-intron endonuclease.        -   3.1.27.10 rRNA endonuclease.    -   3.1.30.-Endoribonucleases active with either ribo- or        deoxyribonucleic producing 5′ phosphomonoesters        -   3.1.30.1 Aspergillus nuclease S(1).        -   3.1.30.2 Serratia marcescens nuclease.    -   3.1.31.-Endoribonucleases active with either ribo- or        deoxyribonucleic producing 3′ phosphomonoesters        -   3.1.31.1 Micrococcal nuclease.

The enzyme is most preferably an exonuclease, such as adeoxyribonuclease, which cleave nucleic acids to form individualnucleotides. The advantages of exodeoxyribonucleases are that they areactive on both single stranded and double stranded DNA and hydrolysebases either in the 5′-3′ or 3′-5′ direction.

An individual nucleotide is a single nucleotide. An individualnucleotide is one which is not bound to another nucleotide or nucleicacid by any bond, such as a phosphodiester bond. A phosphodiester bondinvolves one of the phosphate groups of a nucleotide being bound to thesugar group of another nucleotide. An individual nucleotide is typicallyone which is not bound in any manner to another nucleic acid sequence ofat least 5, at least 10, at least 20, at least 50, at least 100, atleast 200, at least 500, at least 1000 or at least 5000 nucleotides.

Preferred enzymes for use in the method include exonuclease I from E.coli (SEQ ID NO: 6) and RecJ from T. thermophilus (SEQ ID NO: 8) andvariants thereof. The exonuclease enzyme preferably comprises any of thesequences shown in SEQ ID NOs: 6 and 8 or a variant thereof. A variantof SEQ ID NO: 6 or 8 is an enzyme that has an amino acid sequence whichvaries from that of SEQ ID NO: 6 or 8 and which retains nucleic acidhandling ability. The ability of a variant to handle nucleic acids canbe assayed using any method known in the art. For instance, the variantor a pore having the variant attached thereto can be tested for theirability to handle specific sequences of nucleic acids. The enzyme mayinclude modifications that facilitate handling of the nucleic acidand/or facilitate its activity at high salt concentrations and/or roomtemperature. The enzyme may include modifications that facilitatecovalent attachment to or its interaction with the pore or pore subunit.As discussed above, accessible cysteines may be removed from the enzymeto avoid non-specific reactions with a linker. Alternatively, one ormore reactive cysteines may be introduced into the enzyme, for instanceas part of a genetically-fused peptide linker, to facilitate attachmentto the pore or pore subunit.

Variants may differ from SEQ ID NO: 6 or 8 to the same extent asvariants of SEQ ID NO: 2 differ from SEQ ID NO: 2 or 4 as discussedabove.

A variant of SEQ ID NO: 6 or 8 retains its nucleic acid handlingactivity. A variant typically contains the regions of SEQ ID NO: 6 or 8that are responsible for nucleic acid handling activity. The catalyticdomains of SEQ ID NOs: 6 and 8 are discussed above. A variant of SEQ IDNO: 6 or 8 preferably comprises the relevant catalytic domain. A variantSEQ ID NO: 6 or 8 typically includes one or more modifications, such assubstitutions, additions or deletions, outside the relevant catalyticdomain.

Preferred variants of SEQ ID NO: 6 or 8 are described in a co-pendingapplication being filed simultaneously with this application [J A Kemp &Co Ref: N.106566; Oxford Nanolabs Ref: ONL IP 007] which is incorporatedherein by reference. All the teachings of that application may beapplied equally to the present invention.

Preferred enzymes that are capable of pushing or pulling the constructthrough the pore include polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. The polymerase is preferably a memberof any of the Enzyme Classification (EC) groups 2.7.7.6, 2.7.7.7,2.7.7.19, 2.7.7.48 and 2.7.7.49. The polymerase is preferably aDNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, aDNA-dependent RNA polymerase or an RNA-dependent RNA polymerase. Thehelicase is preferably a member of any of the Enzyme Classification (EC)groups 3.6.1.- and 2.7.7.-. The helicase is preferably an ATP-dependentDNA helicase (EC group 3.6.1.8), an ATP-dependent RNA helicase (EC group3.6.1.8) or an ATP-independent RNA helicase. The topoisomerase ispreferably a member of any of the Enzyme Classification (EC) groups5.99.1.2 and 5.99.1.3.

The enzyme may be labelled with a revealing label. The revealing labelmay be any of those described above.

The enzyme may be isolated from an enzyme-producing organism, such as E.coli, T thermophilus or bacteriophage, or made synthetically or byrecombinant means. For example, the enzyme may be synthesised by invitro translation and transcription as described above and below. Theenzyme may be produced in large scale following purification asdescribed above.

Covalent Attachment of the Enzyme to the Pore

In order to effectively sequence the construct, it is important toensure that a proportion of the nucleotides in the construct isidentified in a successive manner. The fixed nature of the enzyme meansthat a proportion of the nucleotides in the construct affects thecurrent flowing through the pore.

The enzyme attached to the pore handles a construct in such a way that aproportion of the nucleotide in the construct interacts with the pore,preferably the barrel or channel of the pore. Nucleotides are thendistinguished on the basis of the different ways in which they affectthe current flowing through the pore during the interaction.

The fixed nature of the enzyme means that a construct is handled by thepore in a specific manner. For instance, each nucleotide may be digestedfrom one of the construct in a processive manner or the construct may bepushed or pulled through the pore. This ensures that a proportion of thenucleotides in the construct interacts with the pore and is identified.The lack of any interruption in the signal is important when sequencingnucleic acids. In addition, the fixed nature of the enzyme and the poremeans they can be stored together, thereby allowing the production of aready-to-use sensor.

In a preferred embodiment, an exonuclease enzyme, such as adeoxyribonuclease, is attached to the pore such that a proportion of thenucleotides is released from the construct and interacts with the barrelor channel of the pore. In another preferred embodiment, an enzyme thatis capable of pushing or pulling the construct through the pore isattached to the pore such that the construct is pushed or pulled throughthe barrel or channel of the pore and a proportion of the nucleotides inthe construct interacts with the barrel or channel. In this embodiment,the nucleotides may interact with the pore in blocks or groups of morethan one, such as 2, 3 or 4. Suitable enzymes include, but are notlimited to, polymerases, nucleases, helicases and topoisomerases, suchas gyrases. In each embodiment, the enzyme is preferably attached to thepore at a site in close proximity to the opening of the barrel ofchannel of the pore. The enzyme is more preferably attached to the poresuch that its active site is orientated towards the opening of thebarrel of channel of the pore. This means that a proportion of thenucleotides of the construct is fed in the barrel or channel. The enzymeis preferably attached to the cis side of the pore.

The pore is attached to the enzyme. The pore may be attached to theenzyme at more than one, such as two or three, points. Attaching thepore to the enzyme at more than one point can be used to constrain themobility of the enzyme. For instance, multiple attachments may be usedto constrain the freedom of the enzyme to rotate or its ability to moveaway from the pore or pore subunit.

The pore may be in a monomeric form when it is attached to the enzyme(post expression modification). Alternatively, the pore may be anoligomeric pore when it is attached to an enzyme (post oligomerisationmodification).

The pore or pore subunit can be attached to the enzyme using any methodknown in the art. The pore or pore subunit and enzyme may be producedseparately and then attached together. The two components may beattached in any configuration. For instance, they may be attached viatheir terminal (i.e. amino or carboxy terminal) amino acids. Suitableconfigurations include, but are not limited to, the amino terminus ofthe enzyme being attached to the carboxy terminus of the pore or poresubunit and vice versa. Alternatively, the two components may beattached via amino acids within their sequences. For instance, theenzyme may be attached to one or more amino acids in a loop region ofthe pore or pore subunit. In a preferred embodiment, terminal aminoacids of the enzyme are attached to one or more amino acids in the loopregion of a pore or pore subunit. Terminal amino acids and loop regionsare discussed above.

In one preferred embodiment, the pore or pore subunit is geneticallyfused to the enzyme. A pore or pore subunit is genetically fused to anenzyme if the whole construct is expressed from a single polynucleotidesequence. The coding sequences of the pore or pore subunit and enzymemay be combined in any way to form a single polynucleotide sequenceencoding the construct.

The pore or pore subunit and enzyme may be genetically fused in anyconfiguration. The pore or pore subunit and enzyme may be fused viatheir terminal amino acids. For instance, the amino terminus of theenzyme may be fused to the carboxy terminus of the pore or pore subunitand vice versa. The amino acid sequence of the enzyme is preferablyadded in frame into the amino acid sequence of the pore or pore subunit.In other words, the enzyme is preferably inserted within the sequence ofthe pore or pore subunit. In such embodiments, the pore or pore subunitand enzyme are typically attached at two points, i.e. via the amino andcarboxy terminal amino acids of the enzyme. If the enzyme is insertedwithin the sequence of the pore or pore subunit, it is preferred thatthe amino and carboxy terminal amino acids of the enzyme are in closeproximity and are each attached to adjacent amino acids in the sequenceof the pore or pore subunit. In a preferred embodiment, the enzyme isinserted into a loop region of the pore or pore subunit. In anespecially preferred embodiment, the enzyme is inserted between aminoacids, 18 and 19, 44 and 45 or 50 and 51 of SEQ ID NO: 2.

In another preferred embodiment, the pore or pore subunit is chemicallyfused to the enzyme. A pore or pore subunit is chemically fused to anenzyme if the two parts are chemically attached, for instance via alinker molecule. Suitable methods include, but are not limited to,hex-his tag, Ni-NTA, biotin binding to streptavidin, antibody binding toan antigen, primary amine coupling, GST tags binding to glutathione, MBPtags binding to dextrin, Protein A binding to IgG, reaction betweenthiols, nucleic acid hybridization linkers and cysteine linkage. DNAhybridization linkers and cysteine linkage are discussed in more detailbelow. The pore or pore subunit is preferably covalently attached to theenzyme.

The pore must retain its pore forming ability. The pore forming abilityof the pore is typically provided by its α-helices and β-strands.β-barrel pores comprise a barrel or channel that is formed fromβ-strands, whereas α-helix bundle pores comprise a barrel or channelthat is formed from α-helices. The α-helices and β-strands are typicallyconnected by loop regions. In order to avoid affecting the pore formingability, the enzyme is preferably genetically fused to a loop region ofthe pore or pore subunit or inserted into a loop region of the pore orpore subunit. The loop regions of specific subunits are discussed inmore detail above. In a preferred embodiment, enzyme is attached to oneor more of amino acids 8, 9, 17, 18, 19, 44, 45, 50 and 51 of SEQ ID NO:2.

Similarly, the construct retains the nucleic acid handling ability ofthe enzyme, which is also typically provided by its secondary structuralelements (α-helices and β-strands) and tertiary structural elements. Inorder to avoid adversely affecting the nucleic acid handling ability ofthe enzyme, the enzyme is preferably genetically fused to the pore orpore subunit or inserted into the pore or pore subunit via residues orregions that does not affect its secondary or tertiary structure.

The pore or pore subunit may be attached directly to the enzyme. Thepore or pore subunit is preferably attached to the enzyme using one ormore, such as two or three, linkers. The one or more linkers may bedesigned to constrain the mobility of the enzyme. The linkers may beattached to one or more reactive cysteine residues, reactive lysineresidues or non-natural amino acids in the pore, pore subunit subunitand/or enzyme. Suitable linkers are well-known in the art. Suitablelinkers include, but are not limited to, chemical crosslinkers andpeptide linkers. Preferred linkers are amino acid sequences (i.e.peptide linkers) or nucleic acid hybridization linkers. The length,flexibility and hydrophilicity of the peptide or nucleic acidhybridization linkers are typically designed such that it does not todisturb the functions of the pore or pore subunit and enzyme. Preferredflexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10or 16, serine and/or glycine amino acids. More preferred flexiblelinkers include (SG)₁, (SG)₂, (SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S isserine and G is glycine. Preferred rigid linkers are stretches of 2 to30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigidlinkers include (P)₁₂ wherein P is proline.

The nucleic acid hybridization linkers can comprise any of the nucleicacids discussed above. For instance, they may comprise deoxyribonucleicacid (DNA), ribonucleic acid (RNA) or any synthetic nucleic acid knownin the art, such as peptide nucleic acid (PNA), glycerol nucleic acid(GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or othersynthetic polymers with nucleotide side chains. The linkers can also bemodified such they react with one another once they have hybridised.Alternatively, agents may be used to crosslink the linkers once theyhave hybridised to one another.

Preferred nucleic acid hybridization linkers correspond to the first 15,25 or 35 nucleotides from the 5′ end of SEQ ID NO: 10. The linkerpreferably also has TT at the 3′ end to provide extra flexibility. Atthe 3′ end, the linkers have a group, such as maleimide, that allows thelinker to be attached to the nucleic acid binding protein or surface.Maleimide modified oliognucleotides can be obtained commercially, forinstance from ATDBio. More preferred linkers are shown in SEQ ID NOs:11, 12 and 13. Complementary linkers are shown in SEQ ID NOs: 14, 15 and16. SEQ ID NO: 11, 12 or 13 may be attached to one of the nucleic acidbinding protein and surface and the complementary linker (SEQ ID NO: 14,15 or 16 respectively) is attached to the other of the nucleic acidbinding protein and surface. The nucleic acid binding protein andsurface can then be attached together by hybridizing the linkers.

Other preferred chemical crosslinkers are shown in the following Table3.

TABLE 3 Some preferred linkers Name Reacts with Structure 1,4-Bis[3-(2-pyridyldithio)propionamido] butane Thiols

1,11-bis- Maleimidotriethyleneglycol Thiols

3,3′-Dithiodipropionic acid di(N-hydroxysuccinimide ester) Primaryamines

Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) Primaryamines

4,4′- Diisothiocyanatostilbene- 2,2′-disulfonic acid disodium saltPrimary amines

Bis[2-(4- azidosalicylamido)ethyl] disulfide Photo- activated,non-specific

3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester Thiols,primary amines

4-Maleimidobutyric acid N- hydroxysuccinimide ester Thiols, primaryamines

Iodoacetic acid N- hydroxysuccinimide ester Thiols, primary amines

S-Acetylthioglycolic acid N-hydroxysuccinimide ester Thiols, primaryamines

Azide-PEG-maleimide Thiols, alkkyne

Alkyne-PEG-maleimide Thiols, azide

Linkers may be attached to the pore or pore subunit first and then theenzyme, the enzyme first and then the pore or pore subunit or the enzymeand pore or pore subunit at the same time. When the linker is attachedto the pore or pore subunit, it may be a monomeric subunit, part of anoligomer of two or more monomers or part of complete oligomeric pore. Itis preferred that the linker is reacted before any purification step toremove any unbound linker.

A preferred method of attaching the pore or pore subunit to the enzymeis via cysteine linkage. This can be mediated by a bi-functionalchemical linker or by a polypeptide linker with a terminal presentedcysteine residue. α-HL (SEQ ID NO: 2) lacks native cysteine residues sothe introduction of a cysteine into the sequence of SEQ ID NO: 2 enablesthe controlled covalent attachment of the enzyme to the subunit.Cysteines can be introduced at various positions, such as position K8,T9 or N17 of SEQ ID NO: 2 or at the carboxy terminus of SEQ ID NO: 2.The length, reactivity, specificity, rigidity and solubility of anybi-functional linker may be designed to ensure that the enzyme ispositioned correctly in relation to the subunit and the function of boththe subunit and enzyme is retained. Suitable linkers includebismaleimide crosslinkers, such as 1,4-bis(maleimido)butane (BMB) orbis(maleimido)hexane. One draw back of bi-functional linkers is therequirement of the enzyme to contain no further surface accessiblecysteine residues, as binding of the bi-functional linker to thesecannot be controlled and may affect substrate binding or activity. Ifthe enzyme does contain several accessible cysteine residues,modification of the enzyme may be required to remove them while ensuringthe modifications do not affect the folding or activity of the enzyme.In a preferred embodiment, a reactive cysteine is presented on a peptidelinker that is genetically attached to the enzyme. This means thatadditional modifications will not necessarily be needed to remove otheraccessible cysteine residues from the enzyme. The reactivity of cysteineresidues may be enhanced by modification of the adjacent residues, forexample on a peptide linker. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the enzyme or pore or pore subunit, either as a monomer orpart of an oligomer, before a linker is attached.

Cross-linkage of pores, pore subunits or enzymes to themselves may beprevented by keeping the concentration of linker in a vast excess of thepore, pore subunit and/or enzyme. Alternatively, a “lock and key”arrangement may be used in which two linkers are used. For instance,click chemistry, such as azide alkyne Huisgen cycloaddition, may be usedto ensure that the pore or pore subunit only binds to the enzyme and notto itself and vice versa. In a preferred embodiment, theazide-PEG-maleimide and alkyne-PEG-maleimide linkers shown in Table 3above are used. One is attached to the pore or pore subunit and theother is attached to the enzyme. This ensures that binding only occursbetween the pore or pore subunit and the enzyme.

Only one end of each linker may react together to form a longer linkerand the other ends of the linker each react with a different part of theconstruct (i.e. subunit or monomer).

The site of covalent attachment is selected such that the enzyme handlesa construct in such a way that a proportion of the nucleotides in theconstruct interacts with the pore. Nucleotides are then distinguished onthe basis of the different ways in which they affect the current flowingthrough the pore during the interaction.

The enzyme is preferably attached to a part of the pore or pore subunitthat forms part of the cis side of a pore comprising the construct. Inelectrophysiology, the cis side is the grounded side by convention. If ahemolysin pore is inserted correctly into an elcetrophysiologyapparatus, the Cap region is on the cis side. It is well known that,under a positive potential, nucleotides will migrate from the cis to thetrans side of pores used for stochastic sensing. Positioning the enzymeat the cis side of a pore allows it to handle the construct such that aproportion of the nucleotides in the sequence enters the barrel orchannel of the pore and interacts with it. Preferably, at least 20%, atleast 40%, at least 50%, at least 80% or at least 90% of the nucleotidesin the sequence enters the barrel or channel of the pore and interactswith it.

The site and method of covalent attachment is preferably selected suchthat mobility of the enzyme is constrained. This helps to ensure thatthe enzyme handles the construct in such a way that a proportion of thenucleotides in the construct interacts with the pore. For instance,constraining the ability of enzyme to move means that its active sitecan be permanently orientated towards the part of the pore or poresubunit that forms part of the opening of the barrel of channel of thepore. The mobility of the enzyme may be constrained by increasing thenumber of points at which the enzyme is attached to the pore or poresubunit and/or the use of specific linkers.

Molecular Adaptor

In some embodiments, the pore comprises a molecular adaptor thatfacilitates the interaction between the pore and the nucleotides or theconstruct. The presence of the adaptor improves the host-guest chemistryof the pore and nucleotides released from or present in the construct.The principles of host-guest chemistry are well-known in the art. Theadaptor has an effect on the physical or chemical properties of the porethat improves its interaction with nucleotides. The adaptor typicallyalters the charge of the barrel or channel of the pore or specificallyinteracts with or binds to nucleotides thereby facilitating theirinteraction with the pore.

The adaptor mediates the interaction between nucleotides released fromor present in the construct and the pore. The nucleotides preferablyreversibly bind to the pore via or in conjunction with the adaptor. Thenucleotides most preferably reversibly bind to the pore via or inconjunction with the adaptor as they pass through the pore across themembrane. The nucleotides can also reversibly bind to the barrel orchannel of the pore via or in conjunction with the adaptor as they passthrough the pore across the membrane. The adaptor preferably constrictsthe barrel or channel so that it may interact with the nucleotides.

The adaptor is typically cyclic. The adaptor preferably has the samesymmetry as the pore. An adaptor having seven-fold symmetry is typicallyused if the pore is heptameric (e.g. has seven subunits around a centralaxis that contribute 14 strands to a transmembrane β barrel). Likewise,an adaptor having six-fold symmetry is typically used if the pore ishexameric (e.g. has six subunits around a central axis that contribute12 strands to a transmembrane β barrel, or is a 12-stranded β barrel).Any adaptor that facilitates the interaction between the pore and thenucleotide can be used. Suitable adaptors include, but are not limitedto, cyclodextrins, cyclic peptides and cucurbiturils. The adaptor ispreferably a cyclodextrin or a derivative thereof. The adaptor is morepreferably heptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). Table 4 belowshows preferred combinations of pores and adaptors.

TABLE 4 Suitable combinations of pores and adaptors Number of strands inthe transmembrane Pore β-barrel Adaptor Leukocidin 16 γ-cyclodextrin(γ-CD) OmpF 16 γ-cyclodextrin (γ-CD) α-hemolysin (or a variant 14β-cyclodextrin (β-CD) thereof discussed above) 6-monodeoxy-6-monoamino-β-cyclodextrin (am₁β-CD) heptakis-6-amino-β- cyclodextrin(am₇-β-CD) heptakis-(6-deoxy-6- guanidino)-cyclodextrin (gu₇-β-CD) OmpG14 β-cyclodextrin (β-CD) 6-monodeoxy-6- monoamino-β-cyclodextrin(am₁β-CD) heptakis-6-amino-β- cyclodextrin (am₇-β-CD)heptakis-(6-deoxy-6- guanidino)-cyclodextrin (gu₇-β-CD) NalP 12α-cyclodextrin (α-CD) OMPLA 12 α-cyclodextrin (α-CD)

The adaptor is preferably covalently attached to the pore. The adaptorcan be covalently attached to the pore using any method known in theart. The adaptor may be attached directly to the pore. The adaptor ispreferably attached to the pore using a bifunctional crosslinker.Suitable crosslinkers are well-known in the art. Preferred crosslinkersinclude 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate,2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate. The mostpreferred crosslinker is succinimidyl 3-(2-pyridyldithio)propionate(SPDP). Typically, the adaptor is covalently attached to thebifunctional crosslinker before the adaptor/crosslinker complex iscovalently attached to the pore but it is also possible to covalentlyattach the bifunctional crosslinker to the pore before the bifunctionalcrosslinker/pore complex is attached to the adaptor.

The site of covalent attachment is selected such that the adaptorfacilitates interaction of nucleotides released from or present in theconstruct with the pore and thereby allows detection of nucleotides. Forpores based on α-HL, the correct orientation of the adaptor within thebarrel or channel of the pore and the covalent attachment of adaptor tothe pore can be facilitated using specific modifications to SEQ ID NO:2. In particular, every subunit of the pore preferably has a glutamineat position 139 of SEQ ID NO: 2. One or more of the subunits of the poremay have an arginine at position 113 of SEQ ID NO: 2. One or more of thesubunits of the pore may have a cysteine at position 119, 121 or 135 ofSEQ ID NO: 2.

Interaction Between the Pore and Nucleotides

The methods may be carried out using any suitable membrane/pore systemin which a pore having a nucleic acid handling enzyme, such as anexonuclease, attached thereto is inserted into a membrane. The methodsare typically carried out using (i) an artificial membrane comprising apore having a nucleic acid handling enzyme, such as an exonuclease,attached thereto, (ii) an isolated, naturally occurring membranecomprising a pore having a nucleic acid handling enzyme, such as anexonuclease, attached thereto, or (iii) a cell expressing a pore havinga nucleic acid handling enzyme, such as an exonuclease, attachedthereto. The methods are preferably carried out using an artificialmembrane. The membrane may comprise other transmembrane and/orintramembrane proteins as well as other molecules in addition to themodified pore.

The membrane forms a barrier to the flow of ions, nucleotides andnucleic acids. The membrane is preferably a lipid bilayer. Lipidbilayers suitable for use in accordance with the invention can be madeusing methods known in the art. For example, lipid bilayer membranes canbe formed using the method of Montal and Mueller (1972). Lipid bilayerscan also be formed using the method described in InternationalApplication No. PCT/GB08/000563 and PCT/GB07/002856.

The methods of the invention may be carried out using lipid bilayersformed from any membrane lipid including, but not limited to,phospholipids, glycolipids, cholesterol and mixtures thereof. Any of thelipids described in International Application No. PCT/GB08/000563 may beused.

Methods are known in the art for inserting pores into membranes, such aslipid bilayers. Some of those methods are discussed above.

The nucleotide or construct may be contacted with the pore on eitherside of the membrane. The nucleotide or construct may be introduced tothe pore on either side of the membrane. The nucleotide or construct istypically contacted with the side of the membrane on which the enzyme isattached to the pore. This allows the enzyme to handle the constructduring the method.

A proportion of the nucleotides of the construct interacts with the poreand/or adaptor as it passes across the membrane through the barrel orchannel of the pore. Alternatively, if the construct is digested by anexonuclease, the nucleotide may interact with the pore via or inconjunction with the adaptor, dissociate from the pore and remain on thesame side of the membrane. The methods may involve the use of pores inwhich the orientation of the adaptor is fixed. In such embodiments, thenucleotide is preferably contacted with the end of the pore towardswhich the adaptor is oriented. Most preferably, the nucleotide iscontacted with the end of the pore towards which the portion of theadaptor that interacts with the nucleotide is orientated.

The nucleotides may interact with the pore in any manner and at anysite. As discussed above, the nucleotides preferably reversibly bind tothe pore via or in conjunction with the adaptor. The nucleotides mostpreferably reversibly bind to the pore via or in conjunction with theadaptor as they pass through the pore across the membrane. Thenucleotides can also reversibly bind to the barrel or channel of thepore via or in conjunction with the adaptor as they pass through thepore across the membrane.

During the interaction between a nucleotides and the pore, thenucleotide affects the current flowing through the pore in a mannerspecific for that nucleotide. For example, a particular nucleotide willreduce the current flowing through the pore for a particular mean timeperiod and to a particular extent. In other words, the current flowingthrough the pore is distinctive for a particular nucleotide. Controlexperiments may be carried out to determine the effect a particularnucleotide has on the current flowing through the pore. Results fromcarrying out the method of the invention on a test sample can then becompared with those derived from such a control experiment in order toidentify a particular nucleotide.

Apparatus

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore having a nucleicacid handling enzyme attached thereto is inserted into a membrane. Themethods may be carried out using any apparatus that is suitable forstochastic sensing. For example, the apparatus comprises a chambercomprising an aqueous solution and a barrier that separates the chamberinto two sections. The barrier has an aperture in which the membranecontaining the pore is formed. The nucleotide or construct may becontacted with the pore by introducing the nucleic acid into thechamber. The nucleic acid may be introduced into either of the twosections of the chamber, but is preferably introduced into the sectionof the chamber containing the enzyme.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562.

The methods involve measuring the current passing through the poreduring interaction with the nucleotides. Therefore the apparatus alsocomprises an electrical circuit capable of applying a potential andmeasuring an electrical signal across the membrane and pore. The methodsmay be carried out using a patch clamp or a voltage clamp. The methodspreferably involves the use of a voltage clamp.

Conditions

The methods of the invention involve the measuring of a current passingthrough the pore during interaction with nucleotides of a construct.Suitable conditions for measuring ionic currents through transmembranepores are known in the art and disclosed in the Examples. The method iscarried out with a voltage applied across the membrane and pore. Thevoltage used is typically from −400 mV to +400 mV. The voltage used ispreferably in a range having a lower limit selected from −400 mV, −300mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV and an upperlimit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably inthe range 120 mV to 170 mV. It is possible to increase discriminationbetween different nucleotides by a pore of the invention by varying theapplied potential.

The methods are carried out in the presence of any alkali metal chloridesalt. In the exemplary apparatus discussed above, the salt is present inthe aqueous solution in the chamber. Potassium chloride (KCl), sodiumchloride (NaCl) or caesium chloride (CsCl) is typically used. KCl ispreferred. The salt concentration is typically from 0.1 to 2.5M, from0.3 to 1.9M, from 0.5 to 1.8M, from 0.7 to 1.7M, from 0.9 to 1.6M orfrom 1M to 1.4M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.However, lower salt concentrations may have to be used so that theenzyme is capable of functioning.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the methods.One suitable buffer is Tris-HCl buffer. The methods are typicallycarried out at a pH of from 4.0 to 10.0, from 4.5 to 9.5, from 5.0 to9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5.The pH used is preferably about 7.5.

The methods are typically carried out at from 0° C. to 100° C., from 15°C. to 95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C.to 80° C., 19° C. to 70° C., or from 20° C. to 60° C. The methods may becarried out at room temperature. The methods are preferably carried outat a temperature that supports enzyme function, such as about 37° C.Good nucleotide discrimination can be achieved at low saltconcentrations if the temperature is increased. However, lowertemperatures, particularly those below room temperature, result inlonger dwell times and can therefore be used to obtain a higher degreeof accuracy.

In addition to increasing the solution temperature, there are a numberof other strategies that can be employed to increase the conductance ofthe solution, while maintaining conditions that are suitable for enzymeactivity. One such strategy is to use the lipid bilayer to divide twodifferent concentrations of salt solution, a low salt concentration ofsalt on the enzyme side and a higher concentration on the opposite side.One example of this approach is to use 200 mM of KCl on the cis side ofthe membrane and 500 mM KCl in the trans chamber. At these conditions,the conductance through the pore is expected to be roughly equivalent to400 mM KCl under normal conditions, and the enzyme only experiences 200mM if placed on the cis side. Another possible benefit of usingasymmetric salt conditions is the osmotic gradient induced across thepore. This net flow of water could be used to pull nucleotides into thepore for detection. A similar effect can be achieved using a neutralosmolyte, such as sucrose, glycerol or PEG. Another possibility is touse a solution with relatively low levels of KCl and rely on anadditional charge carrying species that is less disruptive to enzymeactivity.

Exonuclease-Based Methods

In one embodiment, the methods of sequencing involve contacting theconstruct with a pore having an exonuclease enzyme, such asdeoxyribonuclease, attached thereto. Any of the exonuclease enzymesdiscussed above may be used in the method. The exonuclease releasesindividual nucleotides from one end of the construct. Exonucleases areenzymes that typically latch onto one end of a nucleic acid sequence anddigest the sequence one nucleotide at a time from that end. Theexonuclease can digest the nucleic acid in the 5′ to 3′ direction or 3′to 5′ direction. The end of the nucleic acid to which the exonucleasebinds is typically determined through the choice of enzyme used and/orusing methods known in the art. Hydroxyl groups or cap structures ateither end of the nucleic acid sequence may typically be used to preventor facilitate the binding of the exonuclease to a particular end of thenucleic acid sequence.

The method involves contacting the construct with the exonuclease sothat the nucleotides are digested from the end of the construct at arate that allows identification of a proportion of nucleotides asdiscussed above. Methods for doing this are well known in the art. Forexample, Edman degradation is used to successively digest single aminoacids from the end of polypeptide such that they may be identified usingHigh Performance Liquid Chromatography (HPLC). A homologous method maybe used in the present invention.

The rate at which the exonuclease can be altered by mutation compared tothe wild type enzyme. A suitable rate of activity of the exonuclease inthe method of sequencing involves digestion of from 0.5 to 1000nucleotides per second, from 0.6 to 500 nucleotides per second, 0.7 to200 nucleotides per second, from 0.8 to 100 nucleotides per second, from0.9 to 50 nucleotides per second or 1 to 20 or 10 nucleotides persecond. The rate is preferably 1, 10, 100, 500 or 1000 nucleotides persecond. A suitable rate of exonuclease activity can be achieved invarious ways. For example, variant exonucleases with a reduced orimproved optimal rate of activity may be used in accordance with theinvention.

Pushing or Pulling DNA Through the Pore

Strand sequencing involves the controlled and stepwise translocation ofnucleic acid polymers through a pore. The majority of DNA handlingenzymes are suitable for use in this application provided theyhydrolyse, polymerise or process single stranded DNA or RNA. Preferredenzymes are polymerases, nucleases, helicases and topoisomerases, suchas gyrases. The enzyme moiety is not required to be in as close aproximity to the pore lumen as for individual nucleotide sequencing asthere is no potential for disorder in the series in which nucleotidesreach the sensing moiety of the pore.

The two strategies for single strand DNA sequencing are thetranslocation of the DNA through the nanopore, both cis to trans andtrans to cis, either with or against an applied potential. The mostadvantageous mechanism for strand sequencing is the controlledtranslocation of single strand DNA through the nanopore with an appliedpotential. Exonucleases that act progressively or processively on doublestranded DNA can be used on the cis side of the pore to feed theremaining single strand through under an applied potential or the transside under a reverse potential. Likewise, a helicase that unwinds thedouble stranded DNA can also be used in a similar manner. There are alsopossibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

The following Example illustrates the invention:

1 EXAMPLE

1.1 Generation of the Sequencing Template

The desired template is generated by the ligation of artificial hairpinadaptors (referred to in this document as “Type I adaptor” and “Type IIadaptor”) to the blunt ends of the double stranded (dsDNA) templatefragments. The adaptors are artificial, chemically synthesised DNAsequences that are designed to facilitate construction, purification andfinal release of the desired single stranded sequencing template. Beingartificial sequences, these adaptors have a great degree of flexibilityin their actual sequence and therefore functionality can be built intothe sequences used.

1.2 Type I Adaptor

The Type I adaptor (FIG. 1) is synthesised as a single stranded DNA(ssDNA) oligonucleotide in which the 5′ terminal nucleotides arecomplementary to the 3′ terminal nucleotides such that under appropriateconditions, an intramolecular hybridisation occurs, generating ablunt-ended ‘hairpin loop’ of DNA with a dsDNA region and a ssDNA‘bubble’ region. The double stranded hybridised region is terminatedwith (for example) a sequence of bases which represent one half of therecognition sequence of a ‘rare cutting’ restriction endonuclease. The‘bubble’ region is a single stranded sequence that can provides ahybridisable ‘hook’ for capture of the structure, and any ligationproducts containing the structure, onto a support surface or bead whichis equipped with the complementary ssDNA sequence. The ‘bubble’ regionmay also contain a sequence that identifies a particular Type I adaptorfrom another otherwise identical Type I adaptor, and thus enables themultiplex analysis of ligation products derived from template DNAs fromdifferent individuals.

1.3 Type II Adaptor

The Type II adaptor (FIG. 2) is not unlike the Type I adaptor in grossstructure, being the product of an intramolecular hybridisation of along oligonucleotide. The structure formed has a terminal end that alsodescribes half of the palindromic rare-cutting restriction enzymepresent at the terminal end of the Type I adaptor hairpin. Additionally,the double stranded region of the Type 11 adaptor contains therecognition sequence of a distinct rare-cutting restriction endonuclease(2^(ry) in FIG. 2). The adaptor may also contain a sequence that can beused to identify the adaptor and is situated between the end describinghalf of the palindromic rare-cutting restriction enzyme (1^(ry) in FIG.2) and the recognition sequence of the distinct rare-cutting restrictionendonuclease (2^(ry) in FIG. 2). The bubble region of single strandedDNA of the Type II adaptor can be markedly smaller than that of the TypeI adaptor, as although it also harbours a selectable marker, this is inthe form of a [Biotin-dT], which enables the capture of any ligationproducts containing a Type II adaptor onto a surface of immobilisedstreptavidin.

1.4 Genomic Template

From high molecular weight genomic template, sequencing template may beprepared in a number of ways. An established method is the randomfragmentation and end repair of the sheared DNA to blunt ends; it is anaccepted and reliable method, and the proposed template generationscheme presumes that this will be the method of choice. However, withmodification, the technique described could be modified to accommodateother methods of fragmentation that generate alternative termini,including ‘sticky’ ends.

1.5 Ligation of Adaptors to Randomly Fragmented and End RepairedTemplate DNA

The fragments of sheared DNA will be equipped with a 5′ PO₄ and a 3′ OHon both strands. Dephosphorylation of the template would preventconcatamerisation of the template fragments, but would present achallenge of then having to repair the nicks left upon ligation of the5′ phosphorylated adaptors. Use of excess concentrations of the adaptorswith phosphorylated template DNA will limit the possibility oftemplate:template ligations, but will mean that a large number ofligation products devoid of inserted template will be created (FIG. 3and FIG. 4).

A variety of different ligation products will be generated by thecombination of Type I, Type II and blunt ended templates:

-   -   Adaptor-Adaptor Products    -   Type I-Type I will not bind to streptavidin and will be        eliminated prior to any RE treatments.    -   Type I-Type II will bind to streptavidin, but will be degraded        by primary RE digestion.    -   Type II-Type I will bind to streptavidin, but will be degraded        by primary RE digestion.    -   Type II-Type II will bind to streptavidin, and may crosslink        streptavidin support beads, but will be degraded by primary RE        digestion.

Adaptor-dsDNA template-adaptor products

-   -   Type I-dsDNA template-Type I will not bind to streptavidin and        will be eliminated prior to any RE treatments.    -   Type I-dsDNA template-Type II will bind to streptavidin, will        survive primary RE digestion and will release the desired        product upon secondary RE digestion.    -   Type II-dsDNA template-Type I will bind to streptavidin, will        survive primary RE digestion and will release the desired        product upon secondary RE digestion.    -   Type II-dsDNA template-Type II will bind to streptavidin, and        may crosslink streptavidin support beads, will survive primary        RE digestion, but will release a ‘single stranded’ template        product (not covalently linked) upon secondary RE digestion.        1.6 Isolation of the Desired Sequencing Template

A strategy for streamlined purification of the desired single strandedproduct is presented (FIG. 5). Post-ligation reaction, all dumbbellstructures incorporating Type II adaptors are captured (by virtue of thebiotin moiety carried on the Type II adaptors) onto an immobilisedstreptavidin surface, and any structure which only contain the Type Iadaptors remain unbound and can be washed away. Treatment of the boundType II adaptor structures with the primary restriction endonucleasewill cleave those bound products formed by the ligation of two adaptorswithout any intervening template DNA. All released fragments can then bewashed away, whereas the desired products are retained bound to theplate. Application of the secondary restriction enzyme will cleave thosebound fragments within the captured Type II adaptor sequence, whetherthe product of the ligation has just one Type II adaptor or both endshave a Type II adaptor. The release products are either the desiredcovalently closed structures (⅔^(rds) of all released structures will bethis form) or will be linearised sequences derived from the TypeII:template:Type II ligation products (⅓^(rd) of the released productswill be this form). The non-closed end of the desired covalently closedstructure will be derived from the Type II adaptor and may contain asequence that may be used to identify that adaptor.

Transferring these released sequences to a fresh plate on which a singlestranded DNA sequence complementary to the sequence of the Type I‘bubble’ will enable capture of only those DNA species derived from aType I:template:Type II ligation product. Washing will remove any otherfragments of DNA and will leave only the desired covalently closedTypeI:template:Type II remnant species, which can then be released fromthe plate (heat, alkali wash) and be denatured ready for exonucleasesequencing.

The above purification scheme has the attraction of being automatable,and in delivering only one species of product: that desired for thesequencing reaction. This product can be released from the immobilisedanti-Type I adaptor bubble plate by a simple alkali wash, after whichthe denatured template DNA (FIG. 6) might be neutralised in the presenceof, for example, a buffer solution containing E. coli single strandedbinding protein, which when bound to the denatured ssDNA will maintainits single stranded form; a prerequisite for maintaining theprocessivity of the E. coli Exonuclease I.

1.7 Exonuclease Sequencing of the Desired Sequencing Template

Upon generation of the desired structure, it will be amenable toexonuclease sequencing, with the exonuclease binding to and digestingthe 3′ end of the single strand. The 5′ monophosphate nucleosidesreleased will be identified in the pore and will give rise to (ideally)a sequence of bases that correspond to, in order (FIG. 7):

-   -   Sequence Start: The sequence of a remnant of the Type II        adaptor, which possibly contains a sequence that may be used to        identify the adaptor.    -   Genomic Sequence: The sequence of a template DNA (on the sense        strand).    -   Type I Common: The sequence of the Type I adaptor (which is also        the ‘capture’ sequence).    -   Type I Identifier: The sequence of the Type I adaptor used to        specifically identify a ligation product in a multiplex        sequencing reaction.    -   Comp. Genomic Sequence: The sequence of the template DNA (on the        antisense strand, so the reverse complement of the sense strand        sequence already generated).    -   Sequence End: The sequence of a remnant of the Type II adaptor        (as the reverse complement of the first bases sequenced), which        possibly contains a sequence that may be used to identify the        adaptor.

Sequence listing SEQ ID NO: 1    1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG  71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA 141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC 211GAAGAAGGTG CTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA 281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA TGAGTACTTT 351AACTTATGGA TTCAACGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCCTTAT TGGTGCAAAT 421GTTTCGATTG GTCATACACT GAAATATGTT CAACCTGATT TCAAAACAAT TTTAGAGAGC CCAACTGATA 491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC 561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC AGCAGATAAC 631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA 701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA 771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGGTACCAAT ACTAAAGATA AATGGACAGA TCGTTCTTCA 841 GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA AT SEQ ID NO: 2    1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE  71EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYMSTLTYGF NGNVTGDDTG KIGGLIGANV 141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR NGSMKAADNF 211LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE 281 RYKIDWEKEE MTN SEQ ID NO: 3    1ATGGCAGATT CTGATATTAA TATTAAAACC GGTACTACAG ATATTGGAAG CAATACTACA GTAAAAACAG  71GTGATTTAGT CACTTATGAT AAAGAAAATG GCATGCACAA AAAAGTATTT TATAGTTTTA TCGATGATAA 141AAATCACAAT AAAAAACTGC TAGTTATTAG AACAAAAGGT ACCATTGCTG GTCAATATAG AGTTTATAGC 211GAAGAAGGTG CTAACAAAAG TGGTTTAGCC TGGCCTTCAG CCTTTAAGGT ACAGTTGCAA CTACCTGATA 281ATGAAGTAGC TCAAATATCT GATTACTATC CAAGAAATTC GATTGATACA AAAGAGTATA GGAGTACTTT 351AACTTATGGA TTCAACGGTA ATGTTACTGG TGATGATACA GGAAAAATTG GCGGCCTTAT TGGTGCACAA 421GTTTCGATTG GTCATACACT GAAATATGTT CAACCTGATT TCAAAACAAT TTTAGAGAGC CCAACTGATA 491AAAAAGTAGG CTGGAAAGTG ATATTTAACA ATATGGTGAA TCAAAATTGG GGACCATACG ATCGAGATTC 561TTGGAACCCG GTATATGGCA ATCAACTTTT CATGAAAACT AGAAATGGTT CTATGAAAGC AGCAGATAAC 631TTCCTTGATC CTAACAAAGC AAGTTCTCTA TTATCTTCAG GGTTTTCACC AGACTTCGCT ACAGTTATTA 701CTATGGATAG AAAAGCATCC AAACAACAAA CAAATATAGA TGTAATATAC GAACGAGTTC GTGATGATTA 771CCAATTGCAT TGGACTTCAA CAAATTGGAA AGGTACCAAT ACTAAAGATA AATGGACAGA TCGTTCTTCA 841 GAAAGATATA AAATCGATTG GGAAAAAGAA GAAATGACAA AT SEQ ID NO: 4    1ADSDINIKTG TTDIGSNTTV KTGDLVTYDK ENGMHKKVFY SFIDDKNHNK KLLVIRTKGT IAGQYRVYSE  71EGANKSGLAW PSAFKVQLQL PDNEVAQISD YYPRNSIDTK EYRSTLTYGF NGNVTGDDTG KIGGLIGAQV 141SIGHTLKYVQ PDFKTILESP TDKKVGWKVI FNNMVNQNWG PYDRDSWNPV YGNQLFMKTR NGSMKAADNF 211LDPNKASSLL SSGFSPDFAT VITMDRKASK QQTNIDVIYE RVRDDYQLHW TSTNWKGTNT KDKWTDRSSE 281 RYKIDWEKEE MTN SEQ ID NO: 5    1ATGATGAATG ACGGTAAGCA ACAATCTACC TTTTTGTTTC ACGATTACGA AACCTTTGGC ACGCACCCCG  71CGTTAGATCG CCCTGCACAG TTCGCAGCCA TTCGCACCGA TAGCGAATTC AATGTCATCG GCGAACCCGA 141AGTCTTTTAC TGCAAGCCCG CTGATGACTA TTTACCCCAG CCAGGAGCCG TATTAATTAC CGGTATTACC 211CCGCAGGAAG CACGGGCGAA AGGAGAAAAC GAAGCCGCGT TTGCCGCCCG TATTCACTCG CTTTTTACCG 281TACCGAAGAC CTGTATTCTG GGCTACAACA ATGTGCGTTT CGACGACGAA GTCACACGCA ACATTTTTTA 351TCGTAATTTC TACGATCCTT ACGCCTGGAG CTGGCAGCAT GATAACTCGC GCTGGGATTT ACTGGATGTT 421ATGCGTGCCT GTTATGCCCT GCGCCCGGAA GGAATAAACT GGCCTGAAAA TGATGACGGT CTACCGAGCT 491TTCGCCTTGA GCATTTAACC AAAGCGAATG GTATTGAACA TAGCAACGCC CACGATGCGA TGGCTGATGT 561GTACGCCACT ATTGCGATGG CAAAGCTGGT AAAAACGCGT CAGCCACGCC TGTTTGATTA TCTCTTTACC 631CATCGTAATA AACACAAACT GATGGCGTTG ATTGATGTTC CGCAGATGAA ACCCCTGGTG CACGTTTCCG 701GAATGTTTGG AGCATGGCGC GGCAATACCA GCTGGGTGGC ACCGCTGGCG TGGCATCCTG AAAATCGCAA 771TGCCGTAATT ATGGTGGATT TGGCAGGAGA CATTTCGCCA TTACTGGAAC TGGATAGCGA CACATTGCGC 841GAGCGTTTAT ATACCGCAAA AACCGATCTT GGCGATAACG CCGCCGTTCC GGTTAAGCTG GTGCATATCA 911ATAAATGTCC GGTGCTGGCC CAGGCGAATA CGCTACGCCC GGAAGATGCC GACCGACTGG GAATTAATCG 981TCAGCATTGC CTCGATAACC TGAAAATTCT GCGTGAAAAT CCGCAAGTGC GCGAAAAAGT GGTGGCGATA1051TTCGCGGAAG CCGAACCGTT TACGCCTTCA GATAACGTGG ATGCACAGCT TTATAACGGC TTTTTCAGTG1121ACGCAGATCG TGCAGCAATG AAAATTGTGC TGGAAACCGA GCCGCGTAAT TTACCGGCAC TGGATATCAC1191TTTTGTTGAT AAACGGATTG AAAAGCTGTT GTTCAATTAT CGGGCACGCA ACTTCCCGGG GACGCTCGAT1261TATGCCGAGC AGCAACGCTG GCTGGAGCAC CGTCGCCAGG TCTTCACGCC AGAGTTTTTG CAGGGTTATG1331CTGATGAATT GCAGATGCTG GTACAACAAT ATGCCGATGA CAAAGAGAAA GTGGCGCTGT TAAAAGCACT1401 TTGGCAGTAC GCGGAAGAGA TTGTC SEQ ID NO: 6    1MMNDGKQQST FLFHDYETFG THPALDRPAQ FAAIRTDSEF NVIGEPEVFY CKPADDYLPQ PGAVLITGIT  71PQEARAKGEN EAAFAARIHS LFTVPKTCIL GYNNVRFDDE VTRNIFYRNF YDPYAWSWQH DNSRWDLLDV 141MRACYALRPE GINWPENDDG LPSFRLEHLT KANGIEHSNA HDAMADVYAT IAMAKLVKTR QPRLFDYLFT 211HRNKHKLMAL IDVPQMKPLV HVSGMFGAWR GNTSWVAPLA WHPENRNAVI MVDLAGDISP LLELDSDTLR 281ERLYTAKTDL GDNAAVPVKL VHINKCPVLA QANTLRPEDA DRLGINRQHC LDNLKILREN PQVREKVVAI 351FAEAEPFTPS DNVDAQLYNG FFSDADRAAM KIVLETEPRN LPALDITFVD KRIEKLLFNY RARNFPGTLD 421 YAEQQRWLEH RRQVFTPEFL QGYADELQML VQQYADDKEK VALLKALWQY AEEIVSEQ ID NO: 7    1ATGTTTCGTC GTAAAGAAGA TCTGGATCCG CCGCTGGCAC TGCTGCCGCT GAAAGGCCTG CGCGAAGCCG  71CCGCACTGCT GGAAGAAGCG CTGCGTCAAG GTAAACGCAT TCGTGTTCAC GGCGACTATG ATGCGGATGG 141CCTGACCGGC ACCGCGATCC TGGTTCGTGG TCTGGCCGCC CTGGGTGCGG ATGTTCATCC GTTTATCCCG 211CACCGCCTGG AAGAAGGCTA TGGTGTCCTG ATGGAACGCG TCCCGGAACA TCTGGAAGCC TCGGACCTGT 281TTCTGACCGT TGACTGCGGC ATTACCAACC ATGCGGAACT GCGCGAACTG CTGGAAAATG GCGTGGAAGT 351CATTGTTACC GATCATCATA CGCCGGGCAA AACGCCGCCG CCGGGTCTGG TCGTGCATCC GGCGCTGACG 421CCGGATCTGA AAGAAAAACC GACCGGCGCA GGCGTGGCGT TTCTGCTGCT GTGGGCACTG CATGAACGCC 491TGGGCCTGCC GCCGCCGCTG GAATACGCGG ACCTGGCAGC CGTTGGCACC ATTGCCGACG TTGCCCCGCT 561GTGGGGTTGG AATCGTGCAC TGGTGAAAGA AGGTCTGGCA CGCATCCCGG CTTCATCTTG GGTGGGCCTG 631CGTCTGCTGG CTGAAGCCGT GGGCTATACC GGCAAAGCGG TCGAAGTCGC TTTCCGCATC GCGCCGCGCA 701TCAATGCGGC TTCCCGCCTG GGCGAAGCGG AAAAAGCCCT GCGCCTGCTG CTGACGGATG ATGCGGCAGA 771AGCTCAGGCG CTGGTCGGCG AACTGCACCG TCTGAACGCC CGTCGTCAGA CCCTGGAAGA AGCGATGCTG 841CGCAAACTGC TGCCGCAGGC CGACCCGGAA GCGAAAGCCA TCGTTCTGCT GGACCCGGAA GGCCATCCGG 911GTGTTATGGG TATTGTGGCC TCTCGCATCC TGGAAGCGAC CCTGCGCCCG GTCTTTCTGG TGGCCCAGGG 981CAAAGGCACC GTGCGTTCGC TGGCTCCGAT TTCCGCCGTC GAAGCACTGC GCAGCGCGGA AGATCTGCTG1051CTGCGTTATG GTGGTCATAA AGAAGCGGCG GGTTTCGCAA TGGATGAAGC GCTGTTTCCG GCGTTCAAAG1121CACGCGTTGA AGCGTATGCC GCACGTTTCC CGGATCCGGT TCGTGAAGTG GCACTGCTGG ATCTGCTGCC1191GGAACCGGGC CTGCTGCCGC AGGTGTTCCG TGAACTGGCA CTGCTGGAAC CGTATGGTGA AGGTAACCCG1261 GAACCGCTGT TCCTG SEQ ID NO: 8    1MFRRKEDLDP PLALLPLKGL REAAALLEEA LRQGKRIRVH GDYDADGLTG TAILVRGLAA LGADVHPFIP  71HRLEEGYGVL MERVPEHLEA SDLFLTVDCG ITNHAELREL LENGVEVIVT DHHTPGKTPP PGLVVHPALT 141PDLKEKPTGA GVAFLLLWAL HERLGLPPPL EYADLAAVGT IADVAPLWGW NRALVKEGLA RIPASSWVGL 211RLLAEAVGYT GKAVEVAFRI APRINAASRL GEAEKALRLL LTDDAAEAQA LVGELHRLNA RRQTLEEAML 281RKLLPQADPE AKAIVLLDPE GHPGVMGIVA SRILEATLRP VELVAQGKGT VRSLAPISAV EALRSAEDLL 351LRYGGHKEAA GFAMDEALFP AFKARVEAYA ARFPDPVREV ALLDLLPEPG LLPQVFRELA LLEPYGEGNP 421 EPLFL SEQ ID NO: 9 TAGGGATAACAGGGTAAT SEQ ID NO: 10TGTGTTCTATGTCTTATTCTTACTTCGTTATTCTTGTCTCTATTCTGTTTATGTTTCTTGTTTGTTASEQ ID NO: 11 TGTGTTCTATGTCTT TT - (CH2)4 - MAL SEQ ID NO: 12TGTGTTCTATGTCTTATTCTTACTT TT - (CH2)4 SEQ ID NO: 13TGTGTTCTATGTCTTATTCTTACTTCGTTATTCTT TT - (CH2)4 - MAL SEQ ID NO: 14AAGACATAGAACACA TT - (CH2)4 - MAL SEQ ID NO: 15AAGTAAGAATAAGACATAGAACACA TT -(CH2)4 - MAL SEQ ID NO: 16AAGAATAACGAAGTAAGAATAAGACATAGAACACA TT - (CH2)4 - MAL

The invention claimed is:
 1. A method of identifying the source oforigin of a template double stranded genomic DNA comprising: (i)providing a plurality of template double stranded genomic DNA fragmentsfrom at least two different organisms in separate containers, the firstcontainer comprising a first plurality of template double strandedgenomic DNA fragments from a first organism and the second containercomprising a second plurality of template double stranded genomic DNAfragments from a second organism, wherein the first plurality oftemplate double stranded genomic DNA fragments each comprises a sensestrand and an antisense strand, and the second plurality of templatedouble stranded genomic DNA fragments each comprises a sense strand andan antisense strand; (ii) ligating a first hairpin adaptor to each endof the sense and antisense strands of each of the first plurality oftemplate double stranded genomic DNA fragments provided in (i) in orderto produce a first plurality of circular nucleic acid sequencingconstructs, wherein the first plurality of circular nucleic acidsequencing constructs are produced without amplification of the firstplurality of template double stranded genomic DNA fragments, whereineach first hairpin adaptor comprises (a) a first double stranded DNAstem region formed by intramolecular hybridisation between complementary5′ and 3′ ends of a first single stranded DNA oligonucleotide and (b) afirst single stranded hairpin loop, wherein the first double strandedDNA stem region comprises a first nucleic acid sequence identifier onthe 5′ end of the first double stranded DNA stem region and a reversecomplement of the first nucleic acid sequence identifier on the 3′ endof the first double stranded DNA stem region, wherein the first nucleicacid sequence identifier and the reverse complement of the first nucleicacid sequence identifier are hybridised to one another; (iii) ligating asecond hairpin adaptor to each end of the sense and antisense strands ofeach of the second plurality of template double stranded genomic DNAfragments provided in (i) in order to produce a second plurality ofcircular nucleic acid sequencing constructs, wherein the secondplurality of circular nucleic acid sequencing constructs are producedwithout amplification of the second plurality of template genomic DNAfragments, wherein each second hairpin adaptor comprises (a) a seconddouble stranded DNA stem region formed by intramolecular hybridisationbetween complementary 5′ and 3′ ends of a second single stranded DNAoligonucleotide and (b) a second single stranded hairpin loop, whereinthe_second double stranded DNA stem region comprises a second nucleicacid sequence identifier on the 5′ end of the second double stranded DNAstem region and a reverse complement of the second nucleic acid sequenceidentifier on the 3′ end of the second double stranded DNA stem region,wherein the second nucleic acid sequence identifier and the reversecomplement of the second nucleic sequence identifier are hybridised toone another, wherein the second nucleic acid sequence identifier isdifferent from the first nucleic acid sequence identifier; (iv) mixingthe first plurality of circular nucleic acid sequencing constructs andthe second plurality of circular nucleic acid sequencing constructsproduced in steps (ii) and (iii) to produce a circular nucleic acidsequencing construct mixture; (v) after producing the circular nucleicacid sequencing construct mixture, performing multiplexed, direct singlemolecule sequencing of the circular nucleic acid sequencing constructmixture such that the first sequence identifier and the reversecomplement of the first sequence identifier are sequenced, and thesecond sequence identifier and the reverse complement of the secondsequence identifier are sequenced; and (vi) identifying the source oforigin of the first plurality of template double stranded genomic DNAfragments and the second plurality of template double stranded genomicDNA fragments based upon identification of the first and second nucleicacid sequence identifiers.
 2. The method of claim 1, wherein the firstplurality of template double stranded genomic DNA fragments from thefirst organism and/or the second plurality of template double strandedgenomic DNA fragments from the second organism are randomly fragmented.3. The method of claim 1, wherein the first plurality of template doublestranded genomic DNA fragments from the first organism and/or the secondplurality of template double stranded genomic DNA fragments from thesecond organism are end repaired.
 4. The method of claim 1, wherein themultiplexed, direct single molecule sequencing comprises fluorescent DNApolymerisation.
 5. The method of claim 1, further comprising (i) priorto step (iv), providing a plurality of template double stranded genomicDNA fragments from a third organism in a third container, a plurality oftemplate double stranded genomic DNA fragments from a fourth organism ina fourth container, and a plurality of template double stranded genomicDNA fragments from a fifth organism in a fifth container, wherein thethird plurality of template double stranded genomic DNA fragments eachcomprises a sense strand and an antisense strand, the fourth pluralityof template double stranded genomic DNA fragments each comprises a sensestrand and an antisense strand, and the fifth plurality of templatedouble stranded genomic DNA fragments each comprises a sense strand andan antisense strand; (ii) ligating a third hairpin adaptor to each endof the sense and antisense strands of each of the third plurality oftemplate double stranded genomic DNA fragments provided in (i) in orderto produce a third plurality of circular nucleic acid sequencingconstructs, wherein the third plurality of circular nucleic acidsequencing constructs are produced without amplification of the thirdplurality of template double stranded genomic DNA fragments, whereineach third hairpin adaptor comprises (a) a third double stranded DNAstem region formed by intramolecular hybridisation between complementary5′ and 3′ ends of a third single stranded DNA oligonucleotide and (b) athird single stranded hairpin loop, wherein the third double strandedDNA stem region comprises a third nucleic acid sequence identifier onthe 5′ end of third double stranded DNA stem region and a reversecomplement of the third nucleic acid sequence identifier on the 3′ endof the third double stranded DNA stem region, wherein the third nucleicacid sequence identifier and the reverse complement of the third nucleicacid sequence identifier are hybridised to one another; (iii) ligating afourth hairpin adaptor to each end of the sense and antisense strands ofeach of the fourth plurality of template double stranded genomic DNAfragments provided in (i) in order to produce a fourth plurality ofcircular nucleic acid sequencing constructs, wherein the fourthplurality of circular nucleic acid sequencing constructs are producedwithout amplification of the fourth plurality of template genomic DNAfragments, wherein each fourth hairpin adaptor comprises (a) a fourthdouble stranded DNA stem region formed by intramolecular hybridisationbetween complementary 5′ and 3′ ends of a fourth single stranded DNAoligonucleotide and (b) a fourth single stranded hairpin loop, whereinthe fourth double stranded DNA stem region comprises a fourth nucleicacid sequence identifier on the 5′ end of the fourth double stranded DNAstem region and a reverse complement of the fourth nucleic acid sequenceidentifier on the 3′ end of the fourth double stranded DNA stem region,wherein the fourth nucleic acid sequence identifier and the reversecomplement of the fourth nucleic sequence identifier are hybridised toone another, (iii) ligating a fifth hairpin adaptor to each end of thesense and antisense strands of each of the fifth plurality of templatedouble stranded genomic DNA fragments provided in (i) in order toproduce a fifth plurality of circular nucleic acid sequencingconstructs, wherein the fifth plurality of circular nucleic acidsequencing constructs are produced without amplification of the fifthplurality of template genomic DNA fragments, wherein each fifth hairpinadaptor comprises (a) a fifth double stranded DNA stem region formed byintramolecular hybridisation between complementary 5′ and 3′ ends of afifth single stranded DNA oligonucleotide and (b) a fifth singlestranded hairpin loop, wherein the fifth double stranded DNA stem regioncomprises a fifth nucleic acid sequence identifier on the 5′ end of thefifth double stranded DNA stem region and a reverse complement of thefifth nucleic acid sequence identifier on the 3′ end of the fifth doublestranded DNA stem region, wherein the fifth nucleic acid sequenceidentifier and the reverse complement of the fifth nucleic sequenceidentifier are hybridised to one another, wherein the first, second,third, fourth, and fifth nucleic acid sequence identifiers are differentfrom each other; (iv) mixing the first plurality of circular nucleicacid sequencing constructs produced in step (ii), the second pluralityof circular nucleic acid sequencing constructs produced in steps (iii),the third plurality of circular nucleic acid sequencing constructsproduced in steps (ii'), the fourth plurality of circular nucleic acidsequencing constructs produced in steps (iii'), and the firth pluralityof circular nucleic acid sequencing constructs produced in steps (iii”),to produce a circular nucleic acid sequencing construct mixture; (v)after producing the circular nucleic acid sequencing construct mixture,performing multiplexed, direct single molecule sequencing of thecircular nucleic acid sequencing construct mixture such that the thirdsequence identifier and the reverse complement of the third sequenceidentifier are sequenced, the fourth sequence identifier and the reversecomplement of the fourth sequence identifier are sequenced and the fifthsequence identifier and the reverse complement of the fifth sequenceidentifier are sequenced; and (vi) identifying the source of origin ofthe third plurality of template double stranded genomic DNA fragments,the fourth plurality of template double stranded genomic DNA fragmentsand the fifth plurality of template double stranded genomic DNAfragments based upon identification of the third, fourth and fifthnucleic acid sequence identifiers.